Opioid peptides: synthesis and biological properties of [(N gamma-glucosyl,N gamma-methoxy)-alpha, gamma-diamino-(S)-butanoyl]4-deltorphin-1-neoglycopeptide and related analogues.

Article abstract of DOI:10.1039/b306142f

The [D-Ala2]deltorphin 1 sequence in which the aspartic acid residue is replaced by the N gamma-OCH3-alpha, gamma-diamino (S) butanoyl residue was synthesized using the Fmoc-chemistry-based solid phase procedure. The resulting deltorphin analogue was chemoselectively glucosylated by reaction with unprotected D-glucose (Glc). The Asn4-, (2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D-galactopyranosyl)-Asn4- and the (2-acetamido-2-deoxy-D-galactopyranosyl)-Asn4-deltorphin I were also prepared for comparison. The affinity of the new compounds for the delta-opioid receptor was expressed by the inhibition constant (Ki) of the binding of the delta-receptor selective ligand [3H]naltrindole (NTI) to rat brain membrane preparations. The in vitro biological activity of the synthetic peptides was compared with that of the mu-opioid receptor agonist dermorphin in guinea pig ileum (GPI) preparations and with that of the delta-opioid receptor agonist deltorphin I in mouse vas deferens (MVD) preparations. The substitution of Asp4 with Asn failed to affect drastically the Ki and IC50 values for delta-sites, suggesting that an electrostatic interaction does not play an essential role in the binding to delta-opioid sites. The steric hindrance of the side chain of the residue in position 4 affects binding to delta-sites. The increase of the Ki value is smaller when the sugar-peptide linkage involves the gamma-nitrogen of the Dab residue in comparison with the Asn amide side chain.

Full text of DOI:10.1039/b306142f

View Online / Journal Homepage / Table of Contents for this issue
Opioid peptides: synthesis and biological properties of
[(N ꢀ-glucosyl,N ꢀ-methoxy)-ꢁ,ꢀ-diamino-(S )-butanoyl]4-
deltorphin-1-neoglycopeptide and related analogues†
Fernando Filira,a Barbara Biondi,a Laura Biondi,a Elisa Giannini,b Marina Gobbo,a
Lucia Negrib and Raniero Rocchi*a
a Department of Organic Chemistry, University of Padova, Institute of Biomolecular Chemistry,
C. N. R., Section of Padova, via Marzolo, 1 - 35131, Padova, Italy
b Department. of Human Physiology and Pharmacology, University “La Sapienza”,
P.le Aldo Moro, 5-00185 Roma, Italy
Received 2nd June 2003, Accepted 14th July 2003
First published as an Advance Article on the web 29th July 2003
The [-Ala2]deltorphin I sequence in which the aspartic acid residue is replaced by the Nγ-OCH3-α,γ-diamino (S)
butanoyl residue was synthesized using the Fmoc-chemistry-based solid phase procedure. The resulting deltorphin
analogue was chemoselectively glucosylated by reaction with unprotected -glucose (Glc). The Asn4-, (2-acetamido-
3,4,6-tri-O-acetyl-2-deoxy-β--galactopyranosyl)-Asn4- and the (2-acetamido-2-deoxy--galactopyranosyl)-Asn4-
deltorphin I were also prepared for comparison. The affinity of the new compounds for the δ-opioid receptor was
expressed by the inhibition constant (Ki) of the binding of the δ-receptor selective ligand [3H]naltrindole (NTI) to rat
brain membrane preparations. The in vitro biological activity of the synthetic peptides was compared with that of the
µ-opioid receptor agonist dermorphin in guinea pig ileum (GPI) preparations and with that of the δ-opioid receptor
agonist deltorphin I in mouse vas deferens (MVD) preparations. The substitution of Asp4 with Asn failed to affect
drastically the Ki and IC50 values for δ-sites, suggesting that an electrostatic interaction does not play an essential role
in the binding to δ-opioid sites. The steric hindrance of the side chain of the residue in position 4 affects binding to
δ-sites. The increase of the Ki value is smaller when the sugar-peptide linkage involves the γ-nitrogen of the Dab
residue in comparison with the Asn amide side chain.
subcutaneous injection in mice. We also reported21,22 the
development of the oxyamino derivatives of α,β-diamino-(S)-
propanoic acid and α,γ-diamino-(S)-butanoic acid (Dab) for
Introduction
Deltorphins are opioid heptapeptides isolated from frog skin
and are highly selective ligands at δ-opioid receptors.1 When
the preparation of analogues of O-glycosylated serine, or
injected into the brain of mice and rats, deltorphin I and
homoserine (Hse), containing peptides.
deltorphin II produce analgesia,2,3 locomotor stimulation4 and
In particular we described the solution synthesis of the
motivational rewarding5 without development of physical
model neoglycopeptides N α-Boc,N β-(β--Glc)-N β-OBzl-α,β-
dependence6 or respiratory depression.7 It is known that the
diamino-(S)-propanoylserine methyl ester22 and of N α-Fmoc-
use of peptides as therapeutic agents meets with some severe
limitations. Intensive efforts have been made in recent years
to develop peptidomimetics displaying pharmacological
properties more favorable than the prototypes with regard to
specificity of action, resistence towards enzymatic degradation,
pharmacokinetics and bioavailability. Glycosylation of peptides
and other potential therapeutic agents is a promising approach
in rational drug design and a number of elegant approaches
phenylalanyl-N γ–(β--Glc)-N γ-OMe-α,β-diamino-(S)-butano-
ylvaline,23 which corresponds to the 3–5 sequence of deltorphin
I in which Asp 4 was replaced by the N γ-glycosylated Dab
residue. The preparation of another model neoglycopeptide
by the chemoselective ligation methodology based on the
oxyamino chemistry has also been recently described.24 This
paper will focus on the synthesis of N α–Fmoc,N γ-Boc,N γ-
OCH3-α,γ-diamino-(S)-butanoic acid (7) from homoserine
for glycosylation of bioactive peptides have been described. The
and its incorporation into the [-Ala2]deltorphin I sequence
synthesis of glycopeptides and neoglycopeptides has been
(position 4) by using the Fmoc-chemistry-based solid phase
extensively reviewed and excellent articles and reviews
peptide synthesis. Chemoselective glucosylation17 of the
summarize the development in the solution and solid phase
resulting deltorphin analogue C yielded the [N γ-OCH3,N γ-
methodologies.8–16
(βGlc)-Dab4]-deltorphin I (F). The Asn4-deltorphin I (B), the
A new type of chemoselective glycosylation by reaction
of unprotected reducing sugars with any derivatized peptide
(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β--glucopyranosyl)-
Asn4-deltorphin I (D), and the (2-acetamido-2-deoxy-β--
containing a N,O-di-substituted hydroxylamine side chain
glucopyranosyl)-Asn4-deltorphin I (E) were also prepared
function (R1–NH–O–R2) has been described.17,18
for comparison. The amino acid sequences of the synthetic
In previous papers19,20 we described the synthesis of some
deltorphin I analogues are shown in Table 1.
dermorphin and deltorphin analogues β-O- and α-C-glycosyl-
ated on the C-terminal amino acid residue and reported
their opioid receptor affinity and their analgesic potency after
Table 1 Deltorphin I analogues
A
B
C
D
E
F
Tyr––Ala–Phe–Asp–Val–Val–Gly–NH2
Tyr––Ala–Phe–Asn-Val–Val–Gly–NH2
Tyr––Ala–Phe–(N γ–OCH3)Dab–Val–Val–Gly–NH2
Tyr––Ala–Phe–[β-GlcNAc(Ac)3]Asn–Val–Val–Gly–NH2
Tyr––Ala–Phe–(β-GlcNAc)Asn–Val–Val–Gly–NH2
Tyr––Ala–Phe–[N γ–OCH3, N γ-(β-Glc)–]Dab–Val–Val–Gly–NH2
† With the exception of -Ala the amino acid residues are of
-configuration. Standard abbreviations for amino acid derivatives
and peptides are according to the suggestions of the IUPAC-IUB
Commission on Biochemical Nomenclature, Eur. J. Biochem., 1984,
138, 9–37 and of J. Pept. Sci. 2003, 9, 1–8.
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
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 0 5 9 – 3 0 6 3
3059

View Online
Table 2 δ-Opioid receptors affinities and biological activities of synthetic peptides
Peptide
Ki,δ/nM, [3H]NTL
GPI (IC50/nM)
MVD (IC50/nM)
Deltorphin, A
4.7 0.6
44.6 5.2
113 10.3
1474 130
1120 105
280.2 23
—
1.9 0.08
16.9 1.23
38 0.29
1321 98
329 27
[Asn4]-deltorphin, B
4688 410
5102 360
4557 320
18535 1500
31790 3200
[N γ-(OCH3)-Dab4]-deltorphin, C
[β-GlcNAc(Ac)3-Asn4]-deltorphin, D
[β-GlcNAc-Asn4]-deltorphin, E
[N γ-OCH3,N γ-(β-Glc)-Dab4]-deltorphin, F
89 6.5
Ki, dissociation constant; the values are the mean of three experiments SEM. δ-Opioid receptors were labeled with [3H]NLT, 0.1 nM; IC50 agonist
concentration that produced 50% inhibition of the electrically evoked twitch; the values are the mean of not less than six experiments SEM.
Scheme 1 Synthesis of N α–Fmoc,N γ–Boc,N γ–OCH3–Dab–OH.
tains µ-receptors. However, since the substitution of Asp4 with
Asn failed to affect drastically the Ki and IC50 values for δ-sites,
Results and discussion
Peptide synthesis
such electrostatic interactions apparently do not play an essen-
tial role in the binding to δ-opioid sites. In addition to charge,
changes in the steric hindrance might affect binding and
peptides C, D, E and F show a significant increase in the Ki and
IC50 values for δ-sites in comparison with A. The increase in
the Ki value is smaller when the sugar-peptide linkage involves
the γ-nitrogen of the Dab residue (peptide F) in comparison
with the Asn amide side chain (peptide E).
The amino acid derivative containing the O-methyl hydroxyl-
amine side chain function, used for preparing the deltorphin
analogue C, was synthesized as depicted in Scheme 1. Fmoc-
Hse-OH (1) was converted into the corresponding benzyl ester
(2) and was oxidized to benzyl 2-fluorenylmethyloxycarbonyl-
3-formylpropanoate (3) by the Swern procedure.25 Reaction of
3 with O-methyl hydroxylamine, followed by reduction, tert-
butyloxycarbonylation and catalytic hydrogenation yielded the
N α–Fmoc-N γ-Boc-N γ-OCH3-Dab-OH (7) which was used for
peptide elongation by the solid phase procedure. Cleavage of
the heptapeptide from the resin and simultaneous removal of
the Boc group were carried out by treatment with aqueous 95%
trifluoroacetic acid (TFA). The crude peptide C was glyco-
sylated17 and purified by semipreparative HPLC yielding
F. Deltorphin analogues A, B, and D were also prepared by the
solid phase procedure. Fmoc-[β-GlcNAc(Ac)3]-Asn-OH was
used for preparing peptide D which was converted into the
(β-GlcNAc)-Asn4-deltorphin I (E) by treatment with hydrazine
hydrate. Analytical characterization of the deltorphin analogues
was carried out by reverse phase HPLC, amino acid analysis
and mass spectrometry.
Experimental
General methods
All chemicals were commercial products of the best grade
available. TFA, N-(3-dimethylaminoisopropyl)-N-ethylcarbo-
diimide (EDC) and N-methylpyrrolidone (NMP) were Fluka
products; acetonitrile (HPLC grade) was supplied by Carlo
Erba. Rink amide MBHA resin, [4-(2Ј,4Ј-dimethoxyphenyl-
Fmoc-aminomethyl)phenoxyacetamidonorleucyl-4-methyl-4-
benzhydrylamine polystyrene] and Fmoc-[β-GlcNAc(Ac)3]Asn-
OH were obtained from Novabiochem. All other chemicals for
the solid phase synthesis were supplied by Advance Chemtech.
Melting points were taken on a Buchi model 150 melting point
apparatus in open capillaries and are not corrected. Optical
rotations were determined, at 25 ЊC, with a Perkin Elmer model
Pharmacological tests
The in vitro biological activity of the synthesized peptides was
tested in two isolated smooth muscle preparations: guinea pig
ileum (GPI) and mouse vas deferens (MVD) rich in µ- and
δ-opioid receptors, respectively.26 δ-Opioid receptor affinities
are expressed by the inhibition constant (Ki) of the binding of
selective radio ligands to mice brain membrane preparations.
The opioid receptor binding sites were selectively labelled using
[3H]naltrindole (NTI) (Table 2). A previous investigation26
showed that the N-terminal tripeptide appears to be essential
for the best fitting of both deltorphin and dermorphin to their
respective receptor sites and that the C-terminal tripeptide
sequence of [-Ala2]-deltorphins appears to be responsible for
their high δ-opioid receptor affinity. Negatively charged side
chains of Asp4 (or Glu4) could be involved in an electrostatic
ligand repulsion with a negatively charged µ-receptor site or
with a negatively charged membrane compartment that con-
241 polarimeter and [α]D values are given in 10Ϫ1deg cm2 gϪ1
.
Amino acid analyses were made with a Carlo Erba model 3A 29
amino acid analyzer equipped with a Perkin Elmer Signa 10
chromatography data station following hydrolysis for 22 h (46 h
for Val) at 110 ЊC, in sealed, evacuated vials in constant boiling
hydrochloric acid. Ascending thin-layer chromatographies
were routinely performed on TLC plates Silica Gel 60, UV254
,
Machery-Nagel, using the following solvent systems: E1:
butan-1-ol–acetic acid–water–acetonitrile (3 : 1 : 1 : 5 by vol.);
E2: dichloromethane–methanol (25 : 1 v/v); E3: light petrol-
eum–ethyl acetate (2 : 1 v/v); E4: light petroleum : ethyl acetate
(1 : 1 v/v); E5: ethyl acetate–butan-1-ol–acetic acid–water
(15 : 3 : 1 : 1 by vol.). Amino acid derivatives and peptides
were visualized by one or more of the following procedures:
ninhydrin, 4,4Ј-tetramethyldiphenylmethane (TDM) reagent27
and UV light. Sugars and sugar-containing products were
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 0 5 9 – 3 0 6 3
3060

View Online
located by spraying the plates with 10% sulfuric acid in ethanol,
followed by heating for 10 min at 100 ЊC. Low pressure gel
chromatographies (Silica Gel 60, 0.040–0.063 mm, Machery-
Nagel, column 260 × 26 mm, flow rate 10 cm3 minϪ1, fraction
volume 30 cm3) were performed with a Buchi 688 chromato-
graphic pump equipped with a Buchi UV/VIS filter photometer
(254 nm) detector (see Experimental for elution conditions).
Analytical HPLC separations were performed on an Aquapore
RP 300 column (220 × 4.6 mm, 7 µm, Brownlee Labs., flow rate
1.5 cm3 minϪ1) using a Perkin Elmer series 3B liquid chromato-
graph equipped with a LC-90 UV detector and LCI-100
integrator. Eluents A (0.1% TFA in 90% aqueous acetonitrile)
and B (0.1% aqueous TFA) were used for preparing binary
gradients (elution conditions: isocratic 10% A for 2 min, linear
gradient 10–90% A in 30 min). TFA-Containing eluents
induced progressive de-glycosylation of peptide F. To prevent
this inconvenience, TFA was replaced by acetic acid in the
eluents used for HPLC separations (eluent AЈ and BЈ). Semi-
preparative HPLC separations (Vydac 300 column, 250 × 20
mm, 10 µm, flow rate 15 cm3 minϪ1) was performed on a
Shimadzu series SLC-6B chromatograph equipped with two
independent pump units model LC-8, a SPD-6A detector and
a C-R6A integrator (eluents and elution conditions as those
used for the analytical separations). Solvents were dried and
freshly distilled and evaporations were carried out under
reduced pressure at 40–45 ЊC, using a rotary evaporator.
Sodium sulfate was used for drying purposes. Yields are based
on the weight of vacuum-dried products.
(3 × 60 cm3). The pH of the aqueous layer was adjusted to 2–3
with 1 M hydrochloric acid and re-extracted with ethyl acetate
(3 × 50 cm3). The organic extracts were combined, washed with
saturated aqueous sodium chloride, dried and evaporated to
dryness. The residue was dissolved in the minimum amount of
ethyl acetate and precipitated with light petroleum. Yield 4.9 g
(85%), single spot by TLC in E1, mp 124–126 ЊC; [α]D Ϫ19.6
(c 1.0, ethanol). Found: C, 67.06; H, 5.87; N, 4.11. C19H19NO5
1
requires: C, 66.85; H, 5.61; N, 4.12%. H NMR (250 MHz,
DMSO-d6): 1.94–1.62 (m, 2H, βCH2 Hse); 3.55–3.35 (m, 2H,
γCH2 Hse); 4.14–4.02 (m, 1H, αCH Hse); 4.35–4.15 (m, 3H,
CH–CH2 Fmoc); 7.48–7.25 (m, 4H aromatics Fmoc); 7.61 (d,
1H, NH Hse, J 8.0 Hz); 7.92–7.67 (m, 4H, aromatics Fmoc). 1H
NMR (250 MHz, CDCl3, at 50 ЊC owing to the low solubility of
the compound): 2.17 (m, 2H, βCH2 Hse); 3.76 (m, 2H, γCH2
Hse); 4.22 (t, CH Fmoc, J 6.8 Hz); 4.70–4.43 (m, 3H, αCH Hse
and CH2 Fmoc); 5.77 (d, 1H, NH Hse, J 7.3 Hz); 7.80–7.27
(m, 8H, aromatics Fmoc). Lactone formation, evidentiated
by the NMR spectra, occurs slowly on standing in cloroform
solution: 58% after 32 h and 95% after 197 h.
Fmoc-Hse-OBzl, 2
An aqueous 20% solution of cesium carbonate (1.7 cm3, 0.34 g,
1.04 mmol) was added to Fmoc-Hse-OH (0.50 g, 1.46 mmol)
dissolved in a mixture of methanol (6.2 cm3) and water (0.6
cm3). The solvent was removed and the reaction mixture was
taken up (twice) with N,N-dimethylformamide (DMF)(3.7 cm3)
and evaporated to dryness. The cesium salt was taken up once
more with DMF (3.7 cm3) and benzyl bromide (0.2 cm3,
1.7 mmol) was added. After 6 h reaction the solvent was
evaporated in vacuo and the residue taken up with water (25
cm3) and extracted with ethyl acetate (2 × 25 cm3). The organic
layers were combined, dried for less than one hour to prevent
lactone formation, and concentrated to a small volume.
Addition of light petroleum gave a precipitate which was
collected and dried. Yield 0.3 g (48%); single spot by TLC in E2;
mp 107–110 ЊC; [α]D Ϫ6.2 (c 0.97, chloroform). Found: C,
72.06; H, 5.87; N, 3.11. C26H25NO5 requires: C, 72.37; H, 5.84;
Proton NMR spectra at 200 and 250 MHz were recorded at
298 K, unless stated otherwise, on Bruker spectrometers (WP
200 SY and AC-250-F, respectively). Sample concentrations
were in the range 8–10 mg cmϪ3 in CDCl3 or DMSO-d6
(99.996%). Chemical shifts (δ) are expressed relative to the
residual signals at 7.26 ppm in CDCl3, and 2.49 ppm in DMSO-
d6. Proton assignements were determined by selective homo-
spin decoupling. Electrospray ionization mass spectrometry
(ESI-MS) was performed on a PerSeptive Biosystem Mariner
instrument. Ionization potential 4200 V, acceleration potential
100 V.
1
N, 3.25%. H NMR (200 MHz, CDCl3): 2.25–2.10 (m, 2H,
βCH2 Hse); 3.80–3.45 (m, 2H, γCH2 Hse); 4.30–4.10 (t, CH
Fmoc, J 6.7 Hz); 4.70–4.30 (m, 3H, αCH Hse and CH2 Fmoc);
5.20 (s, 2H, CH2 benzyl); 5.68 (d, 1H, NH Hse, J 7.7 Hz); 7.80–
7.20 (m, 13H, aromatics Fmoc and benzyl). Lactone formation,
evidentiated by the NMR spectra, occurs on standing in
cloroform solution: 50% after 6 h.
Chemical synthesis
Assembly of peptides A–D on the Advance Chemtech 348Ω
Peptide Synthesizer was performed on a 0.06 mmol scale, by the
FastMoc methodology (HBTU–HOBt–DIEA, single acylation
protocol, 180 min coupling time, NMP as the solvent), starting
with Rink Amide MBHA resin (0.085 g, substitution
0,73 mmol gϪ1). The final peptide resin was N α-deprotected
with 20% piperidine in NMP, thoroughly washed with NMP
and dried. Cleavage from the resin and removal of the side
chain protecting groups were simultaneously achieved on the
peptide resin by treatment with a mixture of trifluoroacetic
acid–water–triisopropylsilane (95: 2.5 : 2.5 by vol) (10 cm3, 3.5 h
at room temperature). The acid solution was concentrated
in vacuo and the peptide was precipitated twice with excess
tert-butylmethyl ether, collected, dried and lyophylized from
water. Peptide E was obtained from peptide D by deacetylation
of the sugar moiety and peptide C was converted into F by
selective glycosylation.17 All products were characterized by
reverse phase analytical HPLC (elution: isocratic 15% A for
3 min, linear gradient 15–45% A in 25 min), amino acid
composition and molecular weight determination.
N-Fmoc-(2-amino-3-formyl)propanoate-OBzl, 3
Anhydrous dimethylsulfoxide (0.62 cm3) was diluted with
anhydrous dichloromethane (DCM) (2.0 cm3) and added in
25 min to a solution of oxalyl chloride (0.37 cm3, 4.37 mmol) in
anhydrous DCM (7.0 cm3), previously cooled at Ϫ78 ЊC and
kept under nitrogen. The temperature of the reaction mixture
was raised to Ϫ60 ЊC, in 20 min, and a solution of Fmoc-Hse-
OBzl (1.25 g, 2.9 mmol) in anhydrous DCM (5.0 cm3) was
slowly added (50 min). The temperature was adjusted to Ϫ45 ЊC
in 30 min. N,N-diisopropylethylamine (3.0 cm3) and anhydrous
DCM (0.5 cm3) were slowly added and the temperature was
raised to 0 ЊC. Precooled (0 ЊC) 1 M hydrochloric acid (12 cm3)
was added, two phases separated and the aqueous phase was
extracted with DCM (3 × 8 cm3). The combined organic layers
were washed with a phosphate buffer (pH 7.0, 3 × 15 cm3),
dried and evaporated to dryness. The resulting oily residue
(1.2 g) was purified by gel chromatography (eluant: n-hexane–
diethyl ether 2 : 1 v/v) . Yield 0.78 g (63%) single spot by TLC in
E3; mp 100–101 ЊC; [α]D ϩ9.3 (c 1.15, chloroform). Found: C,
72.04; H, 5.77; N, 3.11. C26H23NO5 requires: C, 72.71; H, 5.40;
Fmoc-Hse-OH, 1
Fmoc-OSu (5.7 g, 16.9 mmol) was dissolved in acetonitrile
(40 cm3) and added to a stirred solution of Hse (2.0 g, 16.8
mmol) in aqueous 5% sodium hydrogen carbonate (55 cm3)
(TLC monitoring in E1). The organic solvent was evaporated in
vacuo and the mixture was diluted with aqueous 5% sodium
hydrogen carbonate (50 cm3) and extracted with ethyl acetate
1
N, 3.26%. H NMR (200 MHz, CDCl3,): 3.20–3.10 (m, 2H,
βCH2 Hse); 4.20 (t, 1H, CH Fmoc, J 6.9 Hz); 4.40 (m, 2H, CH2
Fmoc); 4.65 (m, 1H, αCH); 5.20 (s, 2H, CH2 benzyl); 5.72 (d,
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 0 5 9 – 3 0 6 3
3061

View Online
1H, NH, J 8.0 Hz); 7.85–7.20 (m, 13H, aromatics Fmoc and
previously flushed with nitrogen, and catalytically hydrogenated
benzyl); 9.7 (s, 1H, H–C᎐O).
over 10% Pd/C . The reaction was monitored by TLC in E5 and
after 90 min the catalyst was removed by filtration and the
filtrate was evaporated to dryness. The residue was taken up
with ethyl acetate (70 cm3) and washed with 5% sodium
hydrogen carbonate (5 × 40 cm3). The aqueous phases were
combined, acidified to pH ∼1.0 by adding solid potassium
hydrogen sulfate and re-extracted with ethyl acetate (2 × 40
cm3). The organic layers were washed with saturated aqueous
sodium chloride, dried and evaporated to dryness. Yield 1.47 g
(89%); mp 49–50 ЊC; single spot by TLC in E5; [α]D ϩ10.9
(c 1.07, chloroform). Found: C, 63.66; H, 6.27; N, 5.85.
᎐
Benzyl Nꢁ-Fmoc-N ꢀ-(methoxy)imino-ꢁ-amino-L-butanoate, 4
O-Methyl hydroxylamine hydrochloride (0.57 g, 6.84 mmol),
which is scarcely soluble in pyridine, was slowly added, in 5
hours, to a pyridine solution (11 cm3) containing benzyl
N-Fmoc-(2-amino-3-formyl)propanoate (2.88 g, 6.71 mmol).
Molecular sieves (4 Å) were added, the reaction mixture was
stirred overnight and evaporated to dryness. The residue was
taken up with ether (50 cm3), washed with water (2 × 40 cm3),
0.1 M potassium hydrogen sulfate (3 × 35 cm3) and saturated
aqueous sodium chloride and dried . Evaporation of the
solvent yielded the title product (2.91 g, 95%); single spot by
TLC in E3; mp 80–81 ЊC; [α]D ϩ4.3 (c 0.97, chloroform).
Found: C, 70.32; H, 5.87; N, 5.98. C27H26N2O5 requires: C,
1
C25H30N2O7 requires: C, 63.82; H, 6.43; N, 5.95%. H NMR
(250 MHz, CDCl3,): 1.49 (s, 9H, Boc); 2.23–2.05 (m, 2H, βCH2
Hse); 3.76–3.45 (m, 2H, γCH2); 3.69 (s, 3H, OCH3); 4.22 (t, 1H,
CH Fmoc, J 6.9 Hz); 4.51–4.30 (m, 3H, αCH, CH2 Fmoc,); 5.70
(d, 1H, NH Fmoc, J 8.0 Hz); 7.81–7.24 (m, 8H, aromatics
Fmoc).
1
70.73; H, 5.71; N, 6.11%. H NMR (200 MHz, CDCl3,): 2.80
(m, 2H, βCH2 Hse); 3.78 (d, 3H, OCH3); 4.70–4.10 (m, 4H,
αCH, CH–CH2 Fmoc); 5.20 (s, 2H, CH2 benzyl); 5.50 (dd, 1H,
NH); 6.65 (m, 1H, H-vinyl); 7.85–7.20 (m, 13H, aromatics
Fmoc and benzyl).
H–Tyr–D–Ala–Phe–Asp–Val–Val–Gly–NH2, A. Yield: 34 mg
(74%); [M ϩ H]ϩ found 769.3799; C37H52N8O10 requires 768.87.
Amino acid ratios: Asp, 1.00; Gly, 1.01; Ala, 0.97; Val, 1.89;
Tyr, 0.97; Phe, 0.98.
Benzyl Nꢁ-Fmoc-N ꢀ-methoxy-ꢁ,ꢀ-diamino-L-butanoate, 5
H–Tyr–D–Ala–Phe–Asn-Val–Val–Gly–NH2, B. Yield: 31 mg
(67%), [M ϩ H]ϩ found 768.4906; C37H53N9O9 requires 767.89.
Amino acid ratios: Asp, 0.98; Gly, 1.00; Ala, 0.99; Val, 1.85;
Tyr, 0.98; Phe, 1.00.
Sodium cyanoborohydride (0.69 g, 10.98 mmol) was slowly
added, in portions, to a solution of 4 (2.90 g, 6.33 mmol) in
glacial acetic acid (40 cm3). The reaction was monitored by
TLC in E3 and after 3 h the mixture was diluted with water (20
cm3), the pH value was adjusted to 8.0 with 1.0 M acetic acid
and the solution was extracted with ethyl acetate (3 × 40 cm3).
The combined organic layers were washed with 0.1 M potas-
sium hydrogen sulfate (40 cm3), 5% sodium hydrogen carbonate
(40 cm3) and saturated aqueous sodium chloride, dried and
evaporated to dryness. Yield 2.90 g (99%); single spot by TLC in
E3; mp 79–80 ЊC. Found: C, 70.06; H, 6.07; N, 6.14. C27 H28
H–Tyr–D–Ala–Phe–(Nꢀ–OCH3)Dab–Val–Val–Gly–NH2, C.
Yield: 40 mg (85%), [M ϩ H]ϩ found 784.4928; C38H57N9O9
requires 783.94. Amino acid ratios: Gly, 0.98; Ala, 0.98; Val,
1.87; Tyr, 0.97; Phe, 1.02, Dab, not determined.
H–Tyr–D–Ala–Phe–[ꢂ-GlcNAc(Ac)3]Asn–Val–Val–Gly–NH2,
D. Yield: 57 mg (86%), [M ϩ H]ϩ found 1097.58; C51H71N10O17
requires 1096.18. Amino acid ratios: Asp, 0.99; Gly, 1.02; Ala,
0.99; Val, 1.91; Tyr, 0.99; Phe, 1.02.
1
N2O5 requires: C, 70.42; H, 6.13; N, 6.08%. H NMR (250
MHz, CDCl3,): 2.31–1.81 (m, 2H, βCH2 Hse); 3.14–2.85
(m, 2H, γCH2); 3.60 (s, 3H, OCH3); 4.21 (t, 1H, CH Fmoc, J 7.7
Hz); 4.40 (d, 2H, CH2 Fmoc); 4.47–4.45 (m, 1H, αCH); 5.19 (s,
2H, CH2 benzyl); 5.96 (d, 1H, NH Fmoc, J 7.5 Hz); 7.81–7.27
(m, 8H, aromatics Fmoc).
H–Tyr–D–Ala–Phe–(ꢂ-GlcNAc)Asn–Val–Val–Gly–NH2, E.
Hydrazine hydrate (0.05 cm3, 1.029 mmol) was added to a
methanolic solution (2 cm3) of D (50 mg, 0.046 mmol). Further
hydrazine was added in portions (0.03 cm3, 0.617 mmol each)
after 5, 20, 30, 45 and 50 h, and the removal of the acetyl
protecting groups was monitored by analytical HPLC (elution:
isocratic 10% A for 2 min, linear gradient 10–90% A in 30 min.).
After 52 h the reaction mixture was diluted with diethyl ether
(15 cm3) and the resulting precipitate was collected by cen-
trifugation, washed with diethyl ether (15 cm3), dissolved in
water and lyophilized. Yield 39 mg ( 87%); single peak by
analytical HPLC; [M ϩ H]ϩ found 971.4353; C45H65N10O14
requires 970.07. Amino acid ratios: Asp, 0.97; Gly, 1.00; Ala,
0.99; Val, 1.94; Tyr, 0.98; Phe, 1.01.
Benzyl Nꢁ-Fmoc-N ꢀ-Boc-N ꢀ-methoxy-ꢁ,ꢀ-diamino-L-
butanoate, 6
Di-tert-butyl dicarbonate (1.37 g, 6.28 mmol) was added, at 40Њ,
to a solution of 5 (2.89 g, 6.28 mmol) in tetrahydrofuran
(25 cm3) and the reaction was monitored by TLC in E3. Further
di-tert-butyl dicarbonate (1.37 g, 6.28 mmol) was added twice,
after 4.5 h and 6.5 h, respectively, and the reaction mixture was
stirred overnight at 45 ЊC. The solvent was evaporated in vacuo
and the residue was taken up with ethyl acetate (50 cm3) and
washed with 0.1 M potassium hydrogen sulfate (2 × 40 cm3), 5%
sodium hydrogen carbonate (2 × 40 cm3) and saturated aqueous
sodium chloride, dried and evaporated to dryness. The crude
product was purified by chromatography on silica gel (260 ×
26 mm column, n-hexane/diethyl ether 3:1 v/v as the eluent).
Yield 2.97 g (84%, oil); single spot by TLC in E4; [α]D Ϫ0.9Њ
(c 1.11, chloroform). Found: C, 68.06; H, 6.77; N, 4.81.
C32H36N2O7 requires: C, 68.55; H, 6.47; N, 4.99. 1H NMR (250
MHz, CDCl3,): 1.48 (s, 9H, Boc); 2.27–1.94 (m, 2H, βCH2 Hse);
3.67–3.40 (m, 2H, γCH2); 3.64 (s, 3H, OCH3); 4.22 (t, 1H, CH
Fmoc, J 7.1 Hz); 4.53–4.28 (m, 3H, CH2 Fmoc, αCH); 5.18 (s,
2H, CH2 benzyl); 5.57 (d, 1H, NH Fmoc, J 8.4 Hz); 7.81–7.20
(m, 8H, aromatics Fmoc).
H–Tyr–D–Ala–Phe–(Nꢀ–OCH3,
Nꢀ–ꢂ-Glc)Dab–Val–Val–
Gly–NH2, F. The heptapeptide analogue C (18 mg, 0.023
mmol) was dissolved in 2 cm3 of DMF–acetic acid mixture
(1 : 1 v/v) and -glucose (5 mg, 0.03 mmol) was added. The
solution was stirred at 45 ЊC and the reaction was monitored by
analytical HPLC (elution: isocratic 15% AЈ for 3 min, linear
gradient 15–45% AЈ in 25 min). Further -glucose (5 mg, 0.03
mmol) was added after 3.5 h and the reaction mixture was kept
under stirring for 6 h. The solvent was removed and the crude
product was dissolved in a water–acetonitrile mixture (8 : 2 v/v)
and purified by semipreparative HPLC (eluent: isocratic 25%
AЈ for 5 min, linear gradient 25–50% AЈ in 25 min). Lyophiliz-
ation of the product containing peak yielded 5 mg (23%) of a
white product; single peak by analytical HPLC; [M ϩ H]ϩ
found 946.51; C44H67N9O14 requires 945.96. Amino acid ratios:
Asp, 0.99; Gly, 0.98; Ala, 1.01; Val, 1.92; Tyr, 0.98; Phe, 1.00.
Nꢁ-Fmoc-N ꢀ-Boc-N ꢀ-methoxy-ꢁ,ꢀ-diamino-L-butanoic acid, 7
Benzyl N α-Fmoc-N γ-Boc-N γ-methoxy-α,γ-diamino--butano-
ate (1.96 g, 3.5 mmol) was dissolved in methanol (20 cm3),
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 0 5 9 – 3 0 6 3
3062

View Online
Receptor binding assays
References
The binding of deltorphin analogues to δ-opioid receptors was
tested on crude membrane preparations from adult male rats
(Wistar, 250–300 g) as previously described.26 The brains were
homogenized in 50 volumes of Tris-HCl buffer (50 mM, pH
7.4, 4 ЊC) using a Kinematica PT 3000 polytron (20 s, speed
16000 rpm). The homogenate was centrifuged at 41000 × g for
20 min at 4 ЊC, pellets were resuspended in buffer (50 volumes)
and incubated at 25 ЊC for 30 min to remove endogenous
opioids. After centrifugation, pellets were resuspended in
buffer, containing 5% glycerol, to give a final w/v of 20 mg cmϪ3
fresh tissue. The affinity to opioid receptors was determined by
competition of unlabelled compounds with the δ-receptor
selective ligand [3H]NTI (0.1 nM; 35 Ci mmolϪ1). Drugs were
assayed in a final volume of 2 cm3 Tris-HCl buffer (50 mM, pH
7.4), at 35 ЊC for 90 min. All experiments were performed in
triplicate and total binding, nonspecific binding (2.5 µM
[3H]NTI) and 14 inhibitor concentrations were determined.
Reagents and membranes were distributed with a robotic
sample processor (Tecan RSP 5000-series). Using a Brandel
M-24 cell harvester, assays were terminated by filtration
through Whatman GF/B filter strips previously soaked in 0.5%
poly(ethylenimine) for one hour. Filters were washed with
ice-cold buffer (3 × 4 cm3) and radioactivity was counted in a
liquid scintillation spectrometer (Betamatic, Kintron). The
inhibition constant (Ki) of the drugs was calculated from
competitive binding curves with the computer program Prism.
Data are presented as the arithmetic mean SEM of four
independent measurements (Table 2).
1 V. Erspamer, P. Melchiorri, G. Falconieri-Erspamer, L. Negri,
R. Corsi, C. Severini, D. Barra, M. Simmaco and G. Kreil, Proc.
Natl. Acad. Sci. U.S.A., 1989, 86, 5188–5192.
2 Q. Jiang, H. I. Mosberg and F. Porreca, Life Sci., 1900, 47, 43–47.
3 L. Negri, G. Improta, R. Lattanzi, R. Potenza, F. Lucchetti and
P. Melchiorri, Br. J. Pharmacol., 1995, 116, 2931–2938.
4 L. Negri, L. Noviello, V. Noviello and F. Angelucci, Eur. J.
Pharmacol., 1991, 209, 163–168.
5 T. Suzuki, M. Tsuji, T. Mori, H. Ikeda, M. Misawa and H. Nagase,
Brain Res., 1997, 744, 327–334.
6 A. Cowan, X. Z. Xhu, H. Mosberg, J. R. Omnaas and F. Porreca,
J. Pharmacol. Exp. Ther., 1988, 246, 950–955.
7 P. Y. Cheng, D. Wu, J. Decena, Y. Soong, S. McCabe and H. H.
Szeto, Eur. J. Pharmacol., 1993, 230, 85–88.
8 Glycopeptides and related compounds: synthesis, analysis and
applications., eds. D. G. Large and C. D. Warren, Marcel Dekker,
Inc., New York, Basel, Hong Kong, 1997.
9 S. Peters, M. Meldal and K. Bock, in Modern Methods in
Carbohydrate Synthesis, eds. H. Khan and R. A. O’Neill, Harwood
Academic Publishers Inc., Amsterdam, 1996, p. 352.
10 G.-J. Boons and R. L. Polt, in Carbohydrate Chemistry, ed.
G.-J. Boons, Blackie Academic and Professional Inc., London, 1998,
p. 223.
11 K. M. Koeller and C.-H. Wong in Glycoscience, eds. B. Fraser-Reid,
K. Tatsuta and J. Thiem, Springer-Verlag, Berlin, Heidelberg, 2001,
vol. III, p. 2306.
12 H. Hojo and Y. Nakahara, Curr. Prot. Pept. Sci., 2000, 1, 23–48.
13 H. Herzner, T. Reipen, M. Schultz and H. Kunz, Chem. Rev., 2000,
100, 4495–4537.
14 S. A. W. Gruner, E. Locardi, E. Lohof and H. Kessler, Chem. Rev.,
2002, 102, 491–514.
15 F. Schweizer, Angew. Chem., Int. Ed., 2002, 41, 230–253.
16 B. G. Davies, Chem. Rev., 2002, 102, 579–601.
17 F. Peri, P. Dumy and M. Mutter, Tetrahedron, 1998, 54, 12269–12278.
18 M. R. Carrasco, R. T. Brown, I. M. Serafimova and O. Silva,
J. Org. Chem., 2003, 68, 195–197.
19 L. Negri, R. Lattanzi, F. Tabacco, B. Scolaro and R. Rocchi,
Br. J. Pharmacol., 1998, 242, 1516–1522.
20 L. Negri, R. Lattanzi, F. Tabacco, I. Orrù, C. Severini, B. Scolaro
and R. Rocchi, J. Med. Chem., 1999, 42, 400–404.
Activity on isolated organ preparations
Preparations of the myoenteric plexus-longitudinal muscle
obtained from male guinea pig ileum (GPI, rich in µ-opioid
receptors) and preparations of mouse vas deferens (MVD, rich
in δ-opioid receptors) were used for field stimulation with
bipolar rectangular pulses of supramaximal voltage.26 Agonists
were evaluated for their ability to inhibit the electrically-evoked
twitch. The biological potency of peptoid analogues was
compared with that of the µ-opioid receptor agonist der-
morphin in GPI preparations and with that of the δ-opioid
receptor agonist deltorphin-I in MVD preparations. The results
are expressed as the IC50 values obtained from concentration-
response curves (Prism) (Table 2).
21 F. Filira, L. Biondi, M. Gobbo, F. Maran and R. Rocchi, in Hellenic
Forum on Bioactive Peptides, ed. P. A. Cordopatis, Creta University
Press, Athens, Greece, 1997, p. 75.
22 F. Filira, L. Biondi, M. Gobbo and R. Rocchi, in 2nd Hellenic Forum
on Bioactive Peptides, ed. P. A. Cordopatis, Typorama, Patras,
Greece, 2001, p. 21.
23 F. Filira, L. Biondi, M. Gobbo and R. Rocchi, in National
Conference: “From Genes to Molecules”, Siena, Italy, February 8–10,
2002, Book of the Abstracts, PC6, p. 38.
24 F. Peri, S. Venturini and F. Nicotra, in Peptides 2002, eds. E. Benedetti
and C. Pedone, Edizioni Ziino, Napoli, Italy, 2002, p. 252.
25 K. Omura and B. Swern, Tetrahedron, 1978, 34, 1651–1660.
26 P. Melchiorri, L. Negri, G. Falconieri-Erspamer, C. Severini,
R. Corsi, M. Soaje, V. Erspamer and D. Barra, Eur. J. Pharmacol.,
1991, 195, 201–207.
27 E. Von Arx, M. Faupel and M. Bugger, J. Chromatogr., 1976, 120,
224–228.
28 F. Filira, L. Biondi, M. Gobbo, E. Giannini, D. Goldin and
R. Rocchi, in Peptides 2002, eds. E. Benedetti and C. Pedone,
Edizioni Ziino, Napoli, Italy, 2002, p. 96.
Acknowledgements
Financial support from the Italian National Research Council
(CNR) and the Italian Ministry of Instruction, University and
Research (MIUR) is gratefully acknowledged. Presented in part
at the 27th European Peptide Symposium, Sorrento, Italy,
August 31–September 7, 2002. See ref. 28.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 0 5 9 – 3 0 6 3
3063