Stability against enzymatic hydrolysis of endomorphin-1 analogues containing β-proline

Article abstract of DOI:10.1039/b301507f

The enantiomer of endomorphin-1 (Tyr-Pro-Trp-PheNH₂) and the analogues containing (S)-or (R)-β-proline have been synthesized, and their affinities towards μ-opioid receptors have been measured. As expected, the incubations of the different peptides with some commercially available enzymes showed that the presence of D-residues gave strong resistance towards digestion. The presence of β-proline alone is sufficient to confer good resistance against the hydrolysis of the biologically strategic Pro-Trp bond.

Full text of DOI:10.1039/b301507f

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Stability against enzymatic hydrolysis of endomorphin-1 analogues
containing ꢀ-proline
Giuliana Cardillo,*a Luca Gentilucci,*a Alessandra Tolomelli,a Maria Calienni,b
Ahmed R. Qasemb and Santi Spampinatob
a Dipartimento di Chimica “G. Ciamician”, Università di Bologna, via Selmi 2, 40126 Bologna,
Italy. E-mail: gentil@ciam.unibo.it. E-mail: cardillo@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 6th February 2003, Accepted 10th March 2003
First published as an Advance Article on the web 27th March 2003
The enantiomer of endomorphin-1 (Tyr-Pro-Trp-PheNH2) and the analogues containing (S)- or (R)-β-proline have
been synthesized, and their affinities towards µ-opioid receptors have been measured. As expected, the incubations of
the different peptides with some commercially available enzymes showed that the presence of -residues gave strong
resistance towards digestion. The presence of β-proline alone is sufficient to confer good resistance against the
hydrolysis of the biologically strategic Pro–Trp bond.
homo-β-amino acids.20 The affinity for the opioid receptors
Introduction
varied depending on the β-amino acid present.
Despite the enormous need for pain control therapies in mod-
Recent studies performed on endomorphin-1 by molecular
ern medicine, there is not at present any efficient alternative to
modeling and two-dimensional NMR in different environ-
the use of morphine and related alkaloids. Unfortunately,
ments, indicated proline to be the key residue that induces the
a prolonged clinical use of morphine leads to well known
other residues to assume the proper spatial orientation for the
undesirable effects.1 Recently, Zadina isolated the endogenous
best ligand–receptor interaction.17,21
peptides endomorphin-1, Tyr-Pro-Trp-PheNH2, and endo-
Moreover, the introduction of modified proline could play
a special role in the peptide stability in vivo, since the activity of
morphin-2, Tyr-Pro-Phe-PheNH2,2 which revealed high affinity
for the µ-opioid receptor,3 similar to morphine, and play an
DPP IV is particularly directed towards the degradation of
important role in analgesia.4 Recent papers support the con-
proline-containing peptides.14
cept that the immunomodulatory,5 cardiovascular, respiratory,
For this reason, we decided to focus our attention on peptide
and analgesic effects of endomorphin-1-like agonists can be
sequences containing β-prolines carrying the carboxylic acid
group in the 2 position of the pyrrolidinic ring. Previously
dissociated.6
One of the most intriguing outcomes of this discovery, is the
we have reported the enhanced resistance towards enzymatic
possibility of developing endomorphins as novel, safer
hydrolysis of endomorphin-1 analogues containing homo-
analgesics.7 The possibility of using native opioid peptides as
proline.20 In the present work, we report the enzymatic stability
analgesics is generally invalidated by their limited resistance
of peptides containing β-proline in comparison to endo-
morphin-1 (5) and its enantiomer 6.
towards enzymatic hydrolysis in vivo. Endomorphin-1 is easily
degraded by peptidases,8 such as dipeptidyl peptidase IV (DPP
IV), which appears to be a major physiological regulator9 for
Results and discussion
some neuropeptides, regulatory peptides, circulating hormones
and chemokines.10 Several of the endomorphin-1 degradation
products have been isolated from the central nervous system.
None of the detected products had an effect on GTP binding,
nor was able to produce analgesia, suggesting that degradation
prevents the biological activity.11
The synthesis of β-proline was performed by means of a modi-
fied version of a described procedure (Scheme 1).22 To prepare
Boc-(S)-β-proline, we started from Cbz-(R)-3-hydroxypyrrol-
idine 123 (Scheme 1). Tosylation under standard conditions
gave 2, which was treated with KCN, giving the 3-cyano deriv-
ative 3 with complete inversion of the configuration. In the
same step the cyano group was hydrolysed and the benzyloxy-
carbonyl group was removed under acidic conditions, and the
resulting amino acid was protected to give Boc-(S)-β-proline 4
(Scheme 1). In a similar way, starting from Boc-(S)-3-hydroxy-
pyrrolidine we obtained Boc-(R)-β-proline.
Besides the concomitant use of peptidase inhibitors,12
a
possible approach is the use of more stable peptide analogues.13
In several cases, however, peptidomimetics displayed long term
toxicity and difficulties in penetrating the blood–brain barrier,14
in particular when massive structural modifications were
introduced.
Generally, the introduction of -amino acids in an opioid
peptide15 gives an increased stability. Indeed, only a few enzymes
capable of hydrolysing peptide bonds involving -amino acids
have been characterised in multicellular organisms.16 A series
of diastereoisomeric endomorphin-117 and endomorphin-218
analogues containing -amino acids have been synthesized and
their potency measured. However, these peptides in general
exhibited poor affinities towards µ-receptors.
Another possibility we explored is the substitution of
α-amino acids with β-amino acids in the peptide sequence.
During the course of a program directed towards the synthesis
of modified endomorphins, we have reported the preparation
of some endomorphin-1 analogues containing β-amino acids,
having the carboxylic acid group shifted to the β position,19 and
Scheme 1 Synthesis of (S)-Boc-β-proline. Reagents: (a) TsCl, Et3N,
DMAP. (b) KCN, DMSO. (c) 6 M HCl. (d) (Boc)2O, Na2CO3.
The synthesis of the peptides 5–9 was performed by way
of a convergent coupling of the amino acids in solution, under
the conventional Boc conditions, and using EDCI–HOBt as
condensing agents (Scheme 2).24 After each coupling, peptides
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 4 9 8 – 1 5 0 2
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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were purified by flash-chromatography over silica gel. Boc
deprotection with HCl in dioxane gave HCl peptide salts,
which were used without purification. Final purification of the
tetrapeptides by semi-preparative reversed-phase HPLC gave
peptides 5–9 as TFA salts (Scheme 2). Purities were determined
to be 94–97% by analytical reversed-phase HPLC.
Under the conditions reported above, we synthesized endo-
morphin-1 (5), its enantiomer 6, the modified endomorphin
containing (R)-β-Pro 7, its enantiomer 8, and the tetrapeptide
containing (S)-β-Pro 9. The synthesis of 7 and 9 has been
reported in a short communication.19
To test the affinities towards µ-opioid receptors, the new
peptides were incubated in rat brain membrane homogenates
containing the receptors, using [3H]-DAMGO {[3H]-(Tyr--
Ala-Gly-MePhe-glyol); MePhe = N-methylphenylalanine} as
the µ-specific radioligand.
The affinities of 7 (Ki: 0.33 nM, IC50: 1.80 nM) and 9 (Ki: 10.4
nM, IC50: 72.0 nM) have been reported.19 The Ki and IC50
values measured for endomorphin-1 (5) (Ki: 0.16 0.02 nM;
IC50: 4.6 0.3 nM; nH: 0.74 0.03) and DAMGO (Ki: 1.60
0.30 nM; IC50: 9.9
0.6 nM; nH: 0.90
0.05) agree with
the literature.12,25 The affinities measured for the modified
peptides26 were in general lower in comparison to the parent
peptide 5. In a similar way to what was observed for endo-
morphin-2,18 from the comparison of 5 and its enantiomer 6
(Ki: 67.0
2.0 nM; IC50: 780
30 nM; nH: 0.90
0.05),
it appears that the substitution of each amino acid with the
corresponding -stereoisomer caused a decrease of affinity.
Interestingly, peptide 8, which differs from 6 in the presence
of -β-proline, has affinity in the nanomolar range (Ki: 3.8
0.2 nM; IC50: 180
5 nM; nH: 1.04
0.05), despite of the
presence of -amino acids.
The stability of endomorphin-1 and of the modified pep-
tides towards enzymatic degradation has been investigated by
measuring their hydrolysis rates in the presence of some com-
mercially available enzymes: α-chymotrypsin (Fig. 1), carboxy-
peptidase-Y27 (Fig. 2), and aminopeptidase-M27 (Fig. 3).
Peptides were dissolved in a buffer pH = 7.4 and incubated at 37
ЊC with each enzyme in parallel. At designated times, mixture
aliquots were analysed by HPLC. Results were collected in
graphs reporting the amount of starting peptide remaining
(area%) vs. time. To appreciate the resistances displayed by the
different peptides, peak areas were normalized to 100 at t = 0
and the curves were superimposed.
The digestions of 5–9 with α-chymotrypsin were followed
over 5–6 h (Fig. 1). After 1 h the remaining amounts of endo-
morphin-1 (5) and 7 were around 20%, while 9 was still around
Fig.
1
Peptide degradation rates upon incubation with α-
chymotrypsin.
Scheme 2 Synthesis of endomorphin-1 (5), of the enantiomer 6, and
of the analogues 7-9. Reagents: (a) EDCI, HOBt, NEt3. (b) HCl–
dioxane. Ind = indolyl.
Fig. 2 Peptide degradation rates upon incubation with carboxy-
peptidase-Y.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 4 9 8 – 1 5 0 2
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on Merck silica gel 60 (230–400 mesh), and solvents were
simply distilled. NMR spectra were recorded with a Gemini
Varian spectrometer at 300 MHz (1H-NMR) and at 75 MHz
(13C-NMR). Chemical shifts are reported as δ values relative to
the solvent peaks (CHCl3 in CDCl3: 7.26 ppm; CH3OH in
CD3OD: 3.34 ppm). Preparative reversed-phase HPLC was per-
formed on a Waters Delta Prep 4000 Millipore instrument, with
a C18 column RP-18 (40–63 µm). The FAB-mass instrument
employed was a Micromass ZMD spectrometer equipped with
single quadrupole analyzer and a Z-spray ionspray source out-
fitted 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
3.3 software running on a Digital Equipment Corp. personal
computer. Nitrogen was used both as desolvation and nebulizer
gas. The desolvation temperature was set at 200 ЊC and capil-
lary voltage at 3.0 kV. Analytical HPLC was performed on a
HP Series 1100 spectrometer, with a HP Hypersil ODS column
(4.6 µm particle size, 100 Å pore diameter, 250 mm), DAD
215.8 nm. Optical rotations were measured by a Perkin-Elmer
343 polarimeter, and are given in 10Ϫ1 deg cm2 gϪ1. GC-MS
Analyses were performed using a Hewlett-Packard series II
5890 GC with a capillary column HP 5–5% Ph-Me-Si, 30 m,
0.25 micron, ID 0.25 mm, coupled with a MS detector HP 5971.
IR was performed using a Nicolet 210 FT-IR spectrometer.
Enzymatic hydrolyses were performed in a MGM Lauda RC20
thermostatic bath. Carboxypeptidase-Y lyophilized powder,
20% protein content, pH 5, 19 units mgϪ1 solid, 100 units mgϪ1
prot.; aminopeptidase-M suspension in 3.5 M (NH4)2SO4 solu-
tion, pH 7.4, 5.1 mg prot. mLϪ1, 29 units mgϪ1; α-chymotripsin,
60 U mgϪ1, were purchased from Sigma. Homogenates were
centrifuged in Beckman J6B and Beckman J2-21 centrifuges.
Radioactivity was measured by liquid scintillation spectrometry
using a Beckman apparatus.
Fig.
3
Peptide degradation rates upon incubation with amino-
peptidase-M.
80%, and 6 and 8 were almost completely intact (>90%). While
the results obtained for 6 and 8 confirm the resistance expected
for peptides containing -residues,15,16 the resistance displayed
by 9 was less predictable, since the only unusual residue was
(S)-β-Pro.
The degradation of the peptides with carboxypeptidase-Y
under the selected experimental conditions were slower (Fig. 2).
However, after 24 h, endomorphin-1 (5), 7, and 9 were strongly
reduced (around 18–25%), while 8 was still around 90%. Taking
into consideration area errors, with the exception of 8, which
contained all -residues, the other peptides were degraded to
substantially the same extent as endomorphin-1.
The digestions with aminopeptidase-M were monitored over
5–6 h (Fig. 3). During this period, endomorphin-1 (5) was
degraded to approximately 15% of the original amount, for the
cleavage of the Pro–Trp bond.20,27 In contrast, the peptides 6–9
were scarcely degraded, being still present in 85–90%. In par-
ticular, the stabilities displayed by 7 and 9 were of interest, since
the presence of only an (S)-β-proline or an (R)-β-proline
respectively in a sequence of natural residues was sufficient to
ensure a good stability.
(R)-1-Benzyloxycarbonyl-3-tosyloxypyrrolidine ((R)-2)
The study of peptide digestions by aminopeptidase-M could
also be of value to predict the resistance to degradation in vivo.
Indeed, peptides demonstrating a good resistance towards
aminopeptidase-M could also possess stability against the
natural regulator DPP IV, which hydrolyses the same Pro–Trp
bond.28
The comparison of the affinities displayed by the different
peptides for µ-opioid receptors, and the comparison of the
degradation rates in the presence of proteolyic enzymes, indi-
cated β-proline to be the key residue both for biological activity
and resistance to enzymatic hydrolysis.
A mixture of (R)-3-hydroxypyrrolidine hydrochloride (0.50 g,
4.0 mmol), TEA (0.56 mL, 4.0 mmol) and benzyloxycarbonyl
chloride (0.86 mL, 6.0 mmol) was stirred in CH2Cl2 (50 mL) at
0 ЊC. After 2 h solvent was evaporated at reduced pressure, the
residue was diluted with EtOAc and washed with 0.5 M HCl,
with sat. Na2CO3, and dried over Na2SO4. Evaporation of sol-
vent at reduced pressure gave (R)-1 (0.85 g, 95%), used without
further purification. MS m/z 203 (Mϩ Ϫ 18, 17%), 148 (12), 112
(19), 91 (100).
A mixture of (R)-1 (0.85 g, 3.8 mmol), TEA (0.53 mL,
3.8 mmol,), cat. DMAP, and tosyl chloride (0.93 g, 4.9 mmol) in
CH2Cl2 (30 mL), was stirred at 0 ЊC. After 3 h, the mixture was
washed with sat. Na2CO3, and the water layer was extracted
twice with CH2Cl2. Organic layers were collected and dried over
Na2SO4, and solvent was evaporated at reduced pressure. The
resulting crude residue was purified by flash chromatography
over silica gel (eluant: cyclohexane : EtOAc, 80 : 20), giving
pure (R)-2 (1.00 g, 70%). IR: ν 3070, 3050, 1710, 1445, 1370,
1210, 1110 cmϪ1; 1H-NMR (CDCl3) δH 1.98–2.05 (m, 2H), 2.41
(s, 3H), 3.38–3.62 (m, 4H), 5.00–5.09 (m, 1H), 5.10 (s, 2H),
7.30–7.80 (m, 9H); 13C NMR (CDCl3) δC 21.6, 31.4, 32.4, 43.9,
51.8, 67.0, 127.6, 127.8, 127.9, 128.4, 129.9, 136.5, 145.0, 154.0;
MS m/z 272 (2%), 232 (5), 216 (19), 189 (8), 169 (9), 145 (20),
113 (33), 57 (100). [α]D20 = Ϫ11.8 (c 1, CHCl3).
Conclusions
We have synthesized and tested a series of endomorphin
analogues containing β-proline with  or  configuration and/
or -α-amino acids. The modified peptides were more stable
than endomorphin-1 in the presence of proteolytic enzymes. In
particular, 8 remained in all cases almost intact for several
hours under the experimental conditions, while 7 and 9 showed
a good resistance against the hydrolysis of the Pro–Trp bond.
The modifications introduced gave 8 an excellent general resist-
ance, still maintaining an affinity in the nanomolar range.
Therefore, the new modifications introduced could strongly
enhance peptide bioavailability in vivo.
(S )-1-Benzyloxycarbonyl-3-tosyloxypyrrolidine ((S )-2)
Experimental
Under the same reaction conditions as reported for the
synthesis of (R)-1, (S)-3-hydroxypyrrolidine (0.60 g, 4.8 mmol)
gave (S)-1 (1.04 g, 97%). Under the same reaction conditions
as reported for the synthesis of (R)-2, (S)-1 (1.04 g, 4.7 mmol)
was tosylated to give (S)-1 (1.41 g, 80%). [α]D = ϩ12 (c 1,
CHCl3).
General methods
Unless stated otherwise, chemicals were obtained from com-
mercial sources and used without further purification. CH2Cl2
was distilled from P2O5. Flash chromatography was performed
20
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 4 9 8 – 1 5 0 2
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(S )-1-Benzyloxycarbonyl-3-cyanopyrrolidine ((S )-3)
with a C18 column RP-18 (40–63 µm, 250 mm) with a solvent
system A = 0.1% TFA in water and B = 0.1% TFA in
acetonitrile, gradient 100% A to 50% B in 50 min at a 5.0 mL
minϪ1 flow.
To a solution of (R)-2 (1.00 g, 2.7 mmol) in DMSO (15 mL),
KCN (0.35 g, 5.3 mmol) was added and the mixture was stirred
at 90 ЊC for 4 h, then EtOAc was added and the organic layer
was washed three times with small portions of water. The com-
bined water layers were treated with KMnO4 before disposal.
The organic layer was dried over Na2SO4 and solvent was evap-
orated at reduced pressure, giving crude (S)-3, obtained pure
(0.43 g, 70%) after flash chromatography over silica gel (eluant:
cyclohexane : EtOAc, 50 : 50). IR ν 2243, 1704, 1417, 1360,
Purities were determined by analytical reversed-phase HPLC,
under two different systems: HP Hypersil ODS column, 4.6 µm
particle size, 100 Å pore diameter, 250 mm, solvent A = 0.1%
TFA in water and B = 0.1% TFA in acetonitrile, gradient 100%
A to 50% B in 20 min at a 1.0 mL minϪ1 flow, followed by
20 min at 50%; Phenomenex Luna C18 column, 5 µm particle
size, 100 Å pore diameter, 250 mm, solvent A = 0.1% TFA in
water and B = 0.1% TFA in acetonitrile, gradient 90% A to 90%
B in 60 min at 1.0 mL minϪ1 flow. The average purity levels are:
for 5, 97%; for 6, 94%; for 7, 96%; for 8, 96%; for 9, 95%.
1
1195, 696 cmϪ1; H-NMR (CDCl3) δ 2.18–2.38 (m, 2H), 3.02–
3.18 (m, 1H), 3.43–3.81 (m, 4H), 5.20 (s, 2H), 7.30–7.50 (m,
5H); 13C-NMR (CDCl3, major conformer) δ 28.3, 29.3, 44.9,
48.7, 49.1, 67.2, 119.6, 127.9, 128.0, 128.4, 136.2, 154.1; MS m/z
230 (Mϩ, 20%), 185 (3), 123 (7), 91 (100). [α]D20 = ϩ25.4 (c 1.3,
CHCl3).
H-Tyr-Pro-Trp-PheNH2 (5)
1H-NMR (DMSO-d6, major conformer) δH 1.40–1.80 (m, 3H),
1.90–2.02 (m, 1H), 2.70–2.90 (m, 2H), 2.90–3.20 (m, 5H), 3.50–
3.68 (m, 1H), 4.10–4.21 (m, 1H), 4.35–4.58 (m, 3H), 6.80 (d,
J = 7.0 Hz, 2H), 6.90–7.60 (m, 14H), 7.80–7.90 (d, J = 6.7 Hz,
1H), 8.10 (d, J = 6.6 Hz, 1H), 9.40 (s, 1H), 10.80 (s, 1H);
13C-NMR (CD3OD, major conformer) δC 26.0, 28.5, 29.8, 36.9,
38.3, 38.8, 54.5, 55.4, 56.1, 61.6, 110.2, 110.8, 112.5, 116.7,
119.3, 120.0, 122.6, 125.0, 125.3, 125.6, 127.7, 128.7, 129.4,
130.3, 131.4, 131.8, 137.9, 138.0, 158.0, 169.0, 173.4, 175.5.
FAB MS [M ϩ H]: 611.2; calculated for C34H38N6O5: 610.3.
[α]D20 = Ϫ18.5 (c 1, MeOH).
(R)-1-Benzyloxycarbonyl-3-cyanopyrrolidine ((R)-3)
As reported for the synthesis of (S)-3, the reaction of (S)-2
(1.00 g, 2.7 mmol) with KCN gave (R)-3 (0.44 g, 72%). [α]D
Ϫ22.0 (c 0.7, CHCl3).
20
=
(S )-1-(tert-Butoxycarbonyl)pyrrolidine-3-carboxylic acid
((S )-4)
A stirred mixture of (S)-3 (0.43 g, 1.9 mmol), conc. HCl
(10 mL), and conc. CH3COOH (2 mL), was refluxed for 3 h,
then it was cooled at rt. The water layer was washed with Et2O,
and then it was treated with 6 M NaOH at 0 ЊC until pH = 9–10
was reached. The mixture was diluted with acetone (10 mL),
and di-tert-butyl dicarbonate (0.61 g, 2.9 mmol) was added at
0 ЊC. After 3 h, acetone was evaporated at reduced pressure, and
the mixture was treated with 3 M HCl at 0 ЊC until pH = 3 was
reached. The mixture was extracted three times with EtOAc,
and the collected organic layers were dried over Na2SO4. Evap-
oration of the solvent gave (S)-4 (0.35 g, 85%), not needing
further purification. IR ν 3400–3000 br, 1741, 1670, 1429,
H-D-Tyr-D-Pro-D-Trp-D-PheNH2 (6)
20
FAB MS [M ϩ H]: 611.4; calculated for 6: 610.3. [α]D = ϩ17
(c 0.8, MeOH).
H-Tyr-ꢀ-L-Pro-Trp-PheNH2 (7)
1H-NMR (DMSO-d6, major conformer) δH 1.63–1.80 (m, 1H),
1.80–1.99 (m, 1H), 2.70–2.95 (m, 3H), 2.97–3.15 (m, 3H), 3.16–
3.42 (m, 4H), 3.60–3.71 (m, 1H), 4.15–4.20 (m, 1H), 4.40–4.61
(m, 2H), 6.70 (d, J = 7.3 Hz, 2H), 6.90–7.40 (m, 13H), 7.55
(d, J = 6.9 Hz, 1H), 7.87 (d, J = 7.8 Hz, 1H), 8.00 (d, J = 8.0 Hz,
1H), 9.41 (s, 1H), 10.80 (s, 1H); 13C-NMR (CD3OD, major con-
former) δC 27.8, 29.9, 34.0, 36.7, 37.9, 45.2, 49.0, 53.2, 54.5,
67.0, 111.2, 115.6, 118.3, 119.0, 122.6, 124.7, 126.7, 128.3,
129.3, 130.2, 130.5, 137.2, 157.3, 169.0, 171.0, 173.7, 175.0.
FAB MS [M ϩ H]: 611.3; calculated for C34H38N6O5: 610.3.
[α]D20 = Ϫ9 (c 0.2, MeOH).
1
1375, 1167, 1132; H-NMR (CDCl3) δ 1.50 (s, 9H), 2.10–2.20
(m, 2H), 3.00–3.10 (m, 1H), 3.38–3.70 (m, 4H), 10.9 (s, 1H);
13C-NMR (CDCl3) δ 28.2, 28.5, 43.1, 45.1, 47.9, 79.8, 154.4,
177.8. [α]D = ϩ7.7 (c 0.8 CHCl3).
(R)-1-(tert-Butoxycarbonyl)pyrrolidine-3-carboxylic acid
((R)-4)
Hydrolysis of (R)-3 (0.44 g, 1.9 mmol) under acidic conditions
as reported for (S)-3 and protection with di-tert-butyl
dicarbonate gave (R)-4 (0.34 g, 83%). [α]D= Ϫ8.0 (c 0.5 CHCl3).
H-D-Tyr-ꢀ-D-Pro-D-Trp-D-PheNH2 (8)
20
FAB MS [M ϩ H]: 611.6; calculated for 8: 610.3. [α]D = ϩ10
(c 0.2, MeOH).
Synthesis and purification of 5–9
As a general procedure, the peptide coupling was performed
by stirring overnight the HCl salt of the amino amide, the
N-tert-butyloxycarbonyl amino acid (1.0 equiv), triethylamine
(3 equiv), 1-hydroxy-1H-benzotriazole (HOBt; 1.0 equiv), the
HCl salt of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide
(EDCI; 1.2 equiv), in a 9 : 1 mixture of CH2Cl2 and DMF at
0 ЊC. After 12 h, the solvent was evaporated at reduced pressure,
and the mixture was diluted with EtOAc. The organic solution
was in turn washed with 0.5 M HCl, with saturated Na2CO3,
and with brine. The organic layer was dried over Na2SO4, and
solvent was evaporated at reduced pressure. Peptides were
obtained pure by flash-chromatography over silica gel (eluant:
EtOAc : MeOH, 95 : 5) with yields from 60 to 90%.
Boc deprotection was performed by treatment with saturated
HCl in dioxane at 0 ЊC. After 1 h the solvent was evaporated at
reduced pressure and the resulting HCl peptide salts were used
without purification for the next coupling. HCl tetrapeptide
salts were pre-purified by precipitation from MeOH–Et2O.
Final purification was performed by semi-preparative
reversed-phase HPLC on a Waters Delta Prep 4000 Millipore,
H-Tyr-D-ꢀ-Pro-Trp-PheNH2 (9)
1H-NMR (DMSO-d6, major conformer) δH 1.65–1.80 (m, 2H),
2.60–3.00 (m, 4H), 3.10–3.15 (m, 3H), 3.40–3.70 (m, 4H), 4.11–
4.30 (m, 1H), 4.46–4.85 (m, 2H), 6.75 (d, J = 7.1 Hz, 2H), 6.90–
7.60 (m, 14H), 8.00–8.25 (m, 2H), 9.40 (s, 1H), 10.70 (s, 1H);
13C-NMR (CD3OD, major conformer) δC 27.6, 29.8, 36.0, 36.6,
37.5, 37.9, 46.1, 53.2, 54.5, 67.0, 111.3, 115.6, 118.3, 119.0,
123.6, 124.7, 126.7, 128.3, 129.3, 130.2, 130.5, 136.0, 137.2,
157.3, 166.7, 173.1, 174.0, 174.9. FAB MS [M ϩ H]: 611.2;
calculated for C34H38N6O5: 610.3. [α]D20 = ϩ37.9 (c 0.4, MeOH).
Membrane preparations, determination of protein content,
and binding assays
Rat brain, without cerebellum, was weighed and homogenized
in 10 volumes of ice-cold 0.32 M sucrose–10 mM TRIS-HCl
(tris(hydroxymethyl)aminomethane hydrochloride), pH 7.4 at 4
ЊC. The homogenate was centrifuged at 2000 rpm, for 10 min, at
4 ЊC, and the supernatant was in turn centrifuged at 19000 rpm,
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 4 9 8 – 1 5 0 2
1501

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for 20 min, at 4 ЊC. The resulting pellet was suspended in 10
volumes of 50 mM TRIS-HCl–100 mM NaCl (pH 7.4 at 4 ЊC),
as incubation buffer and incubated for 1 h at room temperature
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specific activity was 64 Ci mmolϪ1, Kd = 4.85 nM and Bmax
=
48 fmol mgϪ1 protein; n = 3. Non-specific binding was deter-
mined in the presence of 100 µM DAMGO. The incubation
buffer consisted of 50 mM TRIS-HCl, 0.1% BSA (bovine
serum albumin), pH 7.4 at 4 ЊC, 2 mM EDTA (ethylene-
diaminetetraacetic acid). To prevent any peptidase degradation,
the following protease inhibitors were added to the binding
buffer: captopril (N-[(S)-3-mercapto-2-methylpropionyl]--
proline) 25 µg mLϪ1, bacitracin 0.2 mg mLϪ1, and leupeptin
(N-acetyl--leucyl--leucyl--argininal) 10 µg mLϪ1, phenyl-
methylsulfonyl fluoride 0.19 mg mlϪ1 and aprotinin 5 TIU mlϪ1
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blocked with 0.01 M DADLE ([-Ala2, -Leu5]-enkephalin)
and 0.01 M U50, 488, respectively.
The mixture (1 ml) was incubated for 1 h at room temper-
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and washed with ice cold washing buffer (50 mM TRIS–HCl,
pH 7.4 at 4 ЊC). The ligand–receptor complex radioactivity
retained in the filter was measured by liquid scintillation
spectrometry using a scintillator after 12 h incubation in scintil-
lation cocktail. All assays were performed in triplicate, and
repeated at least three times. Stock solutions (10Ϫ2 M) in
DMSO or MeOH–0.1 M HCl (1 : 1 v/v).
Enzymatic digestion of the tetrapeptides
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diluted with 0.2 mL CH3CN. Sampling intervals were chosen so
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was filtered over PTFE syringe filters, pore diameter 0.20 µm.
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incubation of peptides in TRIS 50, pH 7.4–CH3CN 5 : 2 with
1.5 µL 1 M HCl for 6 h.
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Each hydrolysis experiment was repeated at least twice, and
the reported data are mean values. Error ranges were estimated
on the basis of the standard deviation, and are substantially
similar for the different peptides.
26 Means
standard error (SE) of three experiments performed in
Acknowledgements
triplicate. DAMGO and the tetrapeptides bound to a single class of
receptors (Hill slopes were not significantly different from unity).
P. Taylor and P. A. Insel, in Principles of drug action. The basis of
pharmacology, eds. W. B. Pratt and P. Taylor, Churchill Livingstone,
New York, 1990.
We thank CNR, MURST Cofin 2000 (Programmi di Ricerca
Scientifica di Rilevante Interesse Nazionale) and 60% (Ricerca
Fondamentale Orientata), and the University of Bologna
(funds for selected topics “Sintesi e Caratterizzazione di
Biomolecole per la Salute Umana”) for financial support.
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J. Chromatogr., A, 1999, 846, 39–48.
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P. A. Toth et al., ref. 27. We are inclined to agree with H.-U. Demuth,
who attributed this behaviour to the presence of DPP IV impurities.
29 O. H. Lowry, N. J. Rosenbrough, A. L. Farr and R. J. Randall,
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References and notes
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H. McQuay, S. Mercadante, G. Pasternak and V. Ventafridda,
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 4 9 8 – 1 5 0 2
1502