Synthesis, biological evaluation and structural characterization of novel glycopeptide analogues of nociceptin N/OFQ

Article abstract of DOI:10.1039/c1ob05197k

To examine if the biological activity of the N/OFQ peptide, which is the native ligand of the pain-related and viable drug target NOP receptor, could be modulated by glycosylation and if such effects could be conformationally related, we have synthesized three N/OFQ glycopeptide analogues, namely: [Thr⁵-O-α-d-GalNAc-N/OFQ] (glycopeptide 1), [Ser ¹⁰-O-α-d-GalNAc]-N/OFQ (glycopeptide 2) and [Ser ¹⁰-O-β-d-GlcNAc]-N/OFQ] (glycopeptide 3). They were tested for biological activity in competition binding assays using the zebrafish animal model in which glycopeptide 2 exhibited a slightly improved binding affinity, whereas glycopeptide 1 showed a remarkably reduced binding affinity compared to the parent compound and glycopeptide 3. The structural analysis of these glycopeptides and the parent N/OFQ peptide by NMR and circular dichroism indicated that their aqueous solutions are mainly populated by random coil conformers. However, in membrane mimic environments a certain proportion of the molecules of all these peptides exist as α-helix structures. Interestingly, under these experimental conditions, glycopeptide 1 (glycosylated at Thr-5) exhibited a population of folded hairpin-like geometries. From these facts it is tempting to speculate that nociceptin analogues showing linear helical structures are more complementary and thus interact more efficiently with the native NOP receptor than folded structures, since glycopeptide 1 showed a significantly reduced binding affinity for the NOP receptor.

Full text of DOI:10.1039/c1ob05197k

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Organic &
Biomolecular
Chemistry
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PAPER
Synthesis, biological evaluation and structural characterization of novel
glycopeptide analogues of nociceptin N/OFQ†‡
a
a
b
b
c
Gemma Arsequell, M o` nica Rosa, Carlos Mayato, Rosa L. Dorta, Ver o´ nica Gonzalez-Nunez,
c
d
d
b
c
Katherine Barreto-Valer, Filipa Marcelo, Luis P. Calle, Jes u´ s T. V a´ zquez, Raquel E. Rodr ´ı guez,
Jes u´ s Jim e´ nez-Barbero and Gregorio Valencia*
d
a
Received 4th February 2011, Accepted 31st May 2011
DOI: 10.1039/c1ob05197k
To examine if the biological activity of the N/OFQ peptide, which is the native ligand of the
pain-related and viable drug target NOP receptor, could be modulated by glycosylation and if such
effects could be conformationally related, we have synthesized three N/OFQ glycopeptide analogues,
5
10
namely: [Thr -O-a-D-GalNAc-N/OFQ] (glycopeptide 1), [Ser -O-a-D-GalNAc]-N/OFQ
(
1
0
glycopeptide 2) and [Ser -O-b-D-GlcNAc]-N/OFQ] (glycopeptide 3). They were tested for biological
activity in competition binding assays using the zebrafish animal model in which glycopeptide 2
exhibited a slightly improved binding affinity, whereas glycopeptide 1 showed a remarkably reduced
binding affinity compared to the parent compound and glycopeptide 3. The structural analysis of these
glycopeptides and the parent N/OFQ peptide by NMR and circular dichroism indicated that their
aqueous solutions are mainly populated by random coil conformers. However, in membrane mimic
environments a certain proportion of the molecules of all these peptides exist as a-helix structures.
Interestingly, under these experimental conditions, glycopeptide 1 (glycosylated at Thr-5) exhibited a
population of folded hairpin-like geometries. From these facts it is tempting to speculate that nociceptin
analogues showing linear helical structures are more complementary and thus interact more efficiently
with the native NOP receptor than folded structures, since glycopeptide 1 showed a significantly
reduced binding affinity for the NOP receptor.
Introduction
a
The nociceptin/orphanin FQ peptide (N/OFQ) was the first
peptide discovered by reverse pharmacology. N/OFQ is the
endogenous ligand for the NOP receptor, which belongs to the
Instituto de Qu ´ı mica Avanzada de Catalu n˜ a (IQAC-CSIC), Barcelona,
Spain. E-mail: gregorio.valencia@iqac.csic.es; Fax: +34 93 2045904;
Tel: +34 934006113
1,2
b
Instituto Universitario de Bio-Org a´ nica “Antonio Gonz a´ lez”, Departamento
G-protein-coupled receptor superfamily. Both ligand and receptor
de Qu ´ı mica Org a´ nica, Universidad de La Laguna, La Laguna, Tenerife,
Spain
c
Department of Biochemistry and Molecular Biology, Faculty of Medicine,
bonyl)-L-threonine; Gal, O-b-D-galactopyranose; GalNAc, 2-deoxy-
Instituto de Neurociencias de Castilla y Le o´ n (INCyL), University of
Salamanca, Spain
2
-acetamido-O-b-D-galactopyranosylamine;
GlcNAc,
2-deoxy-2-
acetamido-O-b-D-glucopyranosylamine; HOBt, 1-hydroxybenzotriazole;
HRMS, high resolution mass spectrometry; i.c.v., intracerebroventricular;
N/OFQ, nociceptin/orphanin FQ peptide; [ H]N/OFQ, (leucyl-
3,4,5- H)-nociceptin/orphanin FQ peptide; NMR, nuclear magnetic
d
Chemical and Physical Biology, Centro de Investigaciones Biol o´ gicas (CIB-
CSIC), Madrid, Spain
3
3
†
Electronic supplementary information (ESI) available: HRMS, NMR
and CD data of the N/OFQ and N/OFQ glycopeptide analogues (1–3).
See DOI: 10.1039/c1ob05197k
resonance; NOESY, nuclear Overhauser enhancement spectroscopy;
NOP, nociceptin receptor; Pmc, 2,2,5,7,8-pentamethyl-chroman-6-
sulfonyl; PyBOP, benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium
hexafluorophosphate; rmsd, root-mean-square deviation; RP-HPLC,
reverse phase high-performance liquid chromatography; SAR, structure–
activity relationship; Ser(O-a-D-GlcNAc), O-(2-acetamido-2-deoxy-
a-D-glucopyranosyl)-L-serine; Ser(O-b-D-GlcNAc), O-(2-acetamido-2-
deoxy-b-D-glucopyranosyl)-L-serine; SPPS, solid phase peptide synthesis;
t-Bu, tert-butyl; TFA, trifluoroacetic acid; TFE, trifluoroethanol;
‡
Abbreviations: Boc, tert-butoxycarbonyl; CD, circular dichroism;
CSI, chemical shift indexes; DCM, dichloromethane; DIC, N,N¢-
diisopropylcarbodiimide; DIEA, N,N-diisopropylethylamine; DMEM,
Dulbecco’s Modified Eagle Medium; DMF, N,N-dimethylformamide;
dr: Danio rerio; drNOP, zebrafish NOP receptor; Fmoc, N-fluorenyl-
a
d
9
-yl-methoxycarbonyl; FmocGln(Trt) Wang resin, N -Fmoc-N -trityl-
L-glutamine 4-benzyloxybenzyl ester polymer-bound; Fmoc-Ser[O-a-
D-GalNAc(OAc) ]OH, O-(2-acetamido-2-deoxy-3,4,6-tri-O-acetyl-a-D-
glucopyranosyl)-N-a-(fluoren-9-yl-methoxycarbonyl)-L-serine; Fmoc-
Ser[O-beta-D-GlcNAc(OAc) ]OH, O-(2-acetamido-2-deoxy-3,4,6-tri-O-
acetyl-b-D-glucopyranosyl)-N-a-(fluoren-9-yl-methoxycarbonyl)-L-seri-
ne; Fmoc-Thr[O-a-D-GalNAc(OAc) ]OH, O-(2-acetamido-2-deoxy-
,4,6-tri-O-acetyl-a-D-glucopyranosyl)-N-a-(fluoren-9-yl-methoxycar-
3
Thr(O-a-D-Glc),
O-(a-D-glucopyranosyl)-L-threonine;
Thr(O-a-D-
GlcNAc), O-(2-acetamido-2-deoxy-a-D-glucopyranosyl)-L-threonine;
3
TIS, triisopropylsilane; TOCSY, total correlation spectra; Trt, Trityl.
Abbreviations used for amino acids and designation of peptides follow the
rules of the IUPAC-IUB Commission of Biochemical Nomenclature in J.
Biol. Chem. 1972, 247, 977-983.
3
3
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are widely distributed in the central nervous system and in the
receptors and propeptides, including pronociceptin, and found
that the opioid neurotransmitter system is fully functional and
has been broadly conserved during the course of vertebrate
3,4
spinal cord, but N/OFQ also elicits physiological responses
in the cardiovascular and immune systems, gastrointestinal and
5,6
29
urogenital tracts and airways. Early studies on the central ner-
vous system demonstrated that N/OFQ modulates nociception,
although such effects are hard to interpret and appear to depend on
evolution. The structural analysis of these glycopeptides by
NMR and circular dichroism indicate that glycosylation elicits
conformational changes in N/OFQ that correlate with ligand
affinity.
5
a number of factors, including the route of administration. Thus,
the hyperalgesia initially reported in the hot plate and tail flick
1,2
assays after i.c.v. administration, has since been reclassified as an
Results and discussion
7,8
antianalgesic effect. Besides, i.c.v. administration of this peptide
can functionally antagonize the analgesic effects of morphine and
Design of analogues
5,7,8
other opioids,
whereas N/OFQ has been shown to produce
Two subsequence definitions have been reported in the full
sequence of N/OFQ. A first N-terminal segment (FGGF),
termed the “message”, seems primarily responsible for triggering
stimulation of the receptor. The rest of the sequence, called the
analgesia and/or to potentiate morphine analgesia after intrathe-
5–7,9
cal administration.
Other actions of N/OFQ in the central
nervous system include anxiolytic-like effects, which have been
6,10,11
reported in several behavioural paradigms in animal models.
In addition to this, N/OFQ modulates several neurotransmitter
“
address”, (TGARKSARKLANQ) appears to be involved in
18
12–14
binding and receptor specificity. Another important feature of
N/OFQ is a pharmacophore site that was proposed using NMR
and bioactivity relationship data. The site is defined by the spatial
systems such as glutamate, catecholamines and tachykinins
and is involved in higher brain functions, including learning,
memory, attention, emotion and sensory perception.
30
1
4
8
disposition of residues Phe , Phe and Arg .
Since NOP as well as opioid receptors are G-protein-coupled
receptors, when N/OFQ interacts with its receptor, it activates
several intracellular effectors such as adenylyl cyclase inhibition,
Two obvious O-glycosylation sites in the N/OFQ sequence are
5
10
5
the Thr and Ser positions. Thr is at the hinge between the
“message” and the “address”, joining the two parts together and
2
+
1,2
blockade of Ca channels, and activation of protein kinases and
4
+
6
sitting next to a vertix (Phe ) of the proposed pharmacophore
K channels. However, N/OFQ shows low affinity and negligible
activity at opioid receptors, and opioid ligands have low affinity
1
0
site. On the other hand, Ser , which is located at the “address”,
is flanked by the two identical highly cationic tripeptide clusters
Ala-Arg-Lys. Owing to the highly strategic positions occupied by
15
for the NOP receptor, except for dynorphin A. In addition, NOP
receptors do not bind the opioid antagonist naloxone. For these
reasons, the NOP receptor is currently classified as a non-opioid
member of the opioid receptor family.
5
10
Thr and Ser in the N/OFQ sequence, they were chosen as the
glycosylation points.
The preferred choices for the saccharide part were a and b-, N-
acetylgalactosamine and N-acetylglucosamine as these sugars are
Although the involvement of N/OFQ in such a wide range
of biological functions including pain, cardiovascular control
and immunity may seem an obstacle for the development of
selective drugs, the NOP receptor is currently considered a viable
25
involved in many important biological phenomena. The selected
glycoside and peptide combinations are shown in Fig. 1.
16
drug target. Thus, considerable effort has been invested by the
pharmaceutical industry in developing selective NOP ligands; for
example, the screening of a library of 52 millions of ligands has
led to the identification of five hexapeptides, which are now widely
17
accepted as NOP receptor standards. Additionally, important
contributions to N/OFQ pharmacology have been made by
18–24
academic groups, most notably by G. Calo and R. Guerrini.
A long-standing cooperative research project in our laboratories
has been aimed at the study of pain mechanisms and SAR studies
on opioid ligands. Given that glycosylation causes important
changes in the native conformation, stability, activity and process-
Fig. 1 Structure of the N/OFQ glycopeptides.
25
ing of many proteins, we have been exploring the glycoconjugate
approach for the rational drug design of new opioids. Thus, after
examining the effects exerted by simple sugars on the activity
of some opioids, we have discovered new glycoconjugates with
Glycopeptide 1 was designed to assess the influence of the
presence of a sugar moiety at position 5 and glycopeptides 2 and 3
to test the combined effect of the nature of the sugar (GlcNAc vs.
GalNAc) and the configuration of the glycosidic bond (a vs. b).
26,27
potent antinociceptive properties.
These results are in line with
similar glycoconjugation modifications in other biologically active
28
peptides. As this approach had not yet been applied to the full
native N/OFQ peptide, the aim of this work was to see if the
biological activity of the native N/OFQ ligand could be modulated
by glycosylation and if such effects were conformationally related.
To do so, three different glycosylated N/OFQ analogues and the
parent peptide were prepared. They were tested for biological
activity in competition binding assays using the zebrafish animal
model since we have previously characterized the zebrafish opioid
Synthesis of analogues
The preparation of glycopeptides 1–3 required the synthesis
of three different glycosyl amino acid building blocks suit-
able for solid phase synthesis. The most demanding task
was the preparation of those with a glycosidic bond in
the a-configuration: Fmoc-Ser[O-a-D-GalNAc(OAc)
Fmoc-Thr[O-a-D-GalNAc(OAc) ]OH. This was successfully
3
]OH and
3
6
134 | Org. Biomol. Chem., 2011, 9, 6133–6142
This journal is © The Royal Society of Chemistry 2011

View Article Online
accomplished following the nitroglycal method developed by
residue and glycosidic bond. Most notably, identical modifications
31
32
5
10
Schmidt and improved in our laboratories by the use of methyl
esters as efficient orthogonal carboxylic acid protecting groups and
the introduction of a mild and selective methyl ester deprotection
step effected by lithium iodide. In addition, this protecting group
allowed the nitro group to be reduced with Zn in acid solution,
instead of hydrogen and the unstable platinized RANEYꢀR
(a-GalNAc) at Thr and Ser (glycopeptide 1 vs. glycopeptide 2)
produced opposite effects on the K value of N/OFQ: glycopeptide
2 displayed a slightly lower K value than the parent peptide
N/OFQ, while glycopeptide 1 showed a stronger affinity reduction
i
i
1
0
for the drNOP receptor. In turn, b-GlcNAc glycosylation at Ser
(glycopeptide 3) did not significantly affect ligand affinity. These
results are in accordance with the fact that modifications in the
“address” might modulate peptide affinity. Moreover, the reduc-
tion of affinity observed for glycopeptide 1 may also be related
to the steric disruption of the proposed N/OFQ pharmacophore
nickel. The Fmoc-Ser[O-b-D-GlcNAc(OAc) ]OH building block
3
was prepared by glycosylation of FmocSerOH with peracetylated
GlcNAc using boron trifluoride etherate catalysis according to our
33
own procedures.
1
4
8
4
The three N/OFQ glycopeptides and the control N/OFQ
peptide were manually assembled using these glycosyl amino acid
building blocks by following standard Fmoc protocols of solid
phase peptide synthesis, and were purified by RP-HPLC and
characterized by HRMS.
(Phe /Phe /Arg ) at the Phe position and provide further proof
34
of the important role of this residue in N/OFQ activity.
Studies of N/OFQ glycosylation have been previously con-
ducted only on the truncated and amidated N/OFQ (1–13)-NH
2
35
peptide. This paper describes the synthesis of four O-b-glycosyl
peptides and of two related acetylated intermediates. The two
5
Pharmacological properties of glycosylated nociceptin analogues
truncated Thr glycosylated analogues show far lower biological
activity (IC50 values on the mouse vas deferens preparations)
than the other two glycoconjugates at Ser . These findings are
in agreement with the biological activity reported here for our
native N/OFQ glycopeptides.
The biological characterization of the different nociceptin deriva-
tives was achieved using radioligand binding techniques. The
native nociceptin peptide and the three glycosylated analogues
were tested as unlabelled ligands in competition binding experi-
ments using drNOP membrane homogenates. The four peptides
10
3
Structural studies
were able to effectively displace [ H]-N/OFQ binding, and in all
cases data fitted better to the one-site competition model (Fig.
Synthetic glycosylation of biologically active peptides has often
been sought as a means of improving their pharmacology
and ADME profiles. Given that these objectives are mostly
achieved because sugar residues are able to modulate the bioactive
conformations of peptides, we conducted a set of comparative
structural studies on our N/OFQ glycopeptides to assess a
possible structure–activity relationship (SAR).
2
). According to their K
follows: glycopeptide 2 (K
N/OFQ) (K value = 7.99 ± 1.02 nM) > glycopeptide 3 (K
0.44 ± 2.56 nM) > glycopeptide 1 (K value = 54.95 ± 9.76 nM).
i
values, the rank order of affinity was as
value = 3.81 ± 0.19 nM) > nociceptin
value =
i
(
i
i
1
i
In molecular recognition studies, it is usually assumed that
the bioactive conformation of a given ligand is a well-defined
geometry highly complementary to the receptor binding site. Such
a definition implies that this conformation can only be observed
when the proper ligand–receptor interactions take place. However,
in the case of membrane receptors such as NOP and opioid
receptors it is likely that ligand binding may be preceded by lipid
membrane interaction events like those postulated by membrane
36
compartment theories. Accordingly, the lipid phase of a cellular
membrane may act as a matrix for the receptor as well as the
37
ligand. Theoretical studies of receptor–ligand interactions in
the context of membrane compartmentalization have provided
38
support for this hypothesis, suggesting that the initial interaction
involves the adsorption of the ligand to the lipid membrane. In
turn, such an insertion can induce a specific membrane-bound
conformation that may be close to the bioactive conformation.
For these reasons, we thought it would be of interest to study the
solution conformation of our glycopeptides in membrane mimic
environments using two different techniques. A low dielectric
media, the organic solvent trifluoroethanol (TFE) that promotes
Fig. 2 Pharmacological properties of glycosylated nociceptin analogues.
Competition binding experiments using [ H]-N/OFQ and the glycosy-
lated nociceptins (glycopeptides 1, 2, and 3) together with the parent
peptide on drNOP membrane homogenates. Data were fitted to the
one-site competition model and each point represents the mean ± SEM
3
(
capped bars) of three independent experiments performed in triplicate.
Legend: ꢀ: N/OFQ (K
i
= 7.99 ± 1.02 nM); ꢀ: glycopeptide 1 =
5
[
Thr -O-a-D-GalNAc]-N/OFQ (K
i
= 54.95 ± 9.76 nM); ᭺: glycopeptide
1
0
39
2
= [Ser -O-a-D-GalNAc]-N/OFQ (K
i
= 3.81 ± 0.19 nM); ꢁ: glycopeptide
secondary structure formation, was used to examine the con-
1
0
3
= [Ser -O-b-D-GlcNAc]-N/OFQ (K
i
= 10.44 ± 2.56 nM).
formation of the glycopeptides by circular dichroism. As a more
accurate membrane model for the study of peptide membrane
interactions, SDS micelles were used for conformational studies
These results indicate that glycosylation of N/OFQ has different
39–41
effects on the binding affinity of this endogenous ligand to the
drNOP receptor. In fact, it seems that ligand affinity was affected
not only by the glycosylation site, but also the nature of the glycosyl
of the glycopeptides by NMR techniques.
For comparative
purposes, we also conducted NMR and circular dichroism studies
in water.
This journal is © The Royal Society of Chemistry 2011
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Far-UV circular dichroism studies
Table 1 CD Data of nociceptin and glycosylated nociceptin analogues
and percentage of a-helix (f )
The circular dichroism spectra of the glycopeptides and the parent
N/OFQ in water or phosphate buffered saline solutions (pH
Peptide
[q]195
[q]205
[q]222
f (%)
7
.4) showed a negative Cotton effect just below 200 nm, which
N/OFQ
16572
16333
14243
9762
-11484
-10817
-11375
-6727
-7399
-6681
-7088
-3827
26
24
25
15
is indicative of random structure (Fig. 3A). When these spectra
were recorded at increasing concentrations of TFE to simulate a
more hydrophobic media,
1
2
3
39–41
three Cotton effects were observed,
one positive at 195 and two negative at 205 and 222 nm (Fig.
5
3
B). This pattern is indicative of a secondary structure order and
chemical modification (a-GalNAc) either at Thr (glycopeptide 1)
42
10
characteristic of a-helix.
or Ser (glycopeptide 2) of N/OFQ seems to retain the same
a-helix conformer population as the parent N/OFQ peptide.
Moreover, the CD data obtained for glycopeptide 3 is indicative
of the importance of the stereochemistry of the sugar moiety in
the conformational properties of these glycopeptides.
NMR studies in aqueous solutions
Nociceptin has been structurally elusive and early biophysical
studies have confirmed its tendency not to form well-defined
46
structures in water and other polar solvents. These studies
have also revealed that the N/OFQ secondary structure in
water, as examined by NMR, appears to be random, with rapid
conformational interconversion at NMR time scales. Indeed,
under our experimental conditions (see S.I. Tables S1–S4), the
NMR data for N/OFQ and the glycopeptides (observed NOEs
3
and JHH coupling constants from G6 to Q17) indicated that none
of these compounds show well-defined secondary structures. As
described in S.I. Figures S1–S4, very few long range NOEs could be
30
detected. Also, in good agreement with recent reports, the parent
N/OFQ peptide, particularly its N-terminal part, seems to be
rather flexible. Besides the sequential NOEs, very few inter residual
NOEs could be observed. Those detected at the C-terminus seem
indicative of the presence of a nascent a-helix in water solution.
Fig. 3 3A) CD spectra of nociceptin (black) and glycopeptides 1 (red), 2
blue), and 3 (green) in water. 3B) CD spectra of nociceptin (black) and
glycopeptides 1 (red), 2 (blue), and 3 (green) in TFE.
5
10
Nevertheless, the introduction of a sugar moiety at Thr or Ser
(
produced a slight increase in the number of observed NOEs.
10
Although merely qualitative, glycosylation at Ser permitted a
5
larger number of NOEs to be detected than at Thr , suggesting
In addition, the intensity ratio between the Cotton effects at 205
that this O-Ser substitution induced a certain rigidity of the peptide
chain. On the other hand, no major changes in the chemical shifts
of the different amino acid residues, other than those expected by
glycosylation, were observed. This evidence allowed us to conclude
that besides subtle modifications in the dynamic behaviour of
the peptide chain upon glycosylation, there were no important
structural changes, and that the glycopeptides mainly adopted
disordered conformations in water. This result is in complete
agreement with those obtained by CD analysis.
and 222 nm ([q]222/[q]205) has been associated with the tendency
43
of a given peptide or protein to adopt helical structures. Values
of around 0.8 to 0.95 correspond to compounds with a tendency
to yield high proportions of a-helix. For our glycopeptides and
their parent compound in pure TFE solutions, these values were
around 0.6, which meant their tendency to yield a-helix structures
was low. This data can be further quantified taking into account
that the absorption at 222 nm of a perfect a-helix is up to one order
of magnitude more intense than a random or b-sheet presenting
44
compound. This is why the average ellipticity value observed at
NMR studies in SDS micelles
2
22 nm can be taken as a measure of the proportion of a-helix
in a sample. There are different model equations for the lineal
relationship between the ellipticity of a sample at 222 nm and the
estimated value of a-helix. We used the Luo and Baldwin model
to calculate a-helix proportions (f% values) of around 25% for
N/OFQ and the glycopeptides 1 and 2, and 15% for glycopeptide
As mentioned above, a further aim of this work was to explore
if membrane compartment theories could apply in the case
of N/OFQ and its glycopeptides. Thus, in order to deduce
the possible bioactive conformation of these molecules, NMR
experiments on glycopeptides 1–3 and N/OFQ were conducted,
45
30,47
3
(Table 1), the latter showing 40% less of the secondary ordered
using SDS micelles as mimics of membrane-like environments
structure than the former. This reduction implies that the differ-
ence of nearly one order of magnitude in binding affinity between
glycopeptides 1 and 2 cannot be solely explained in terms of the
relative presence of a-helix conformers. Nevertheless, the same
(see S.I. Tables S5–S8). Under these conditions, a significant
increase in the number of NOE cross peaks, especially arising from
NH–NH contacts, was observed for all tested compounds (see
S.I. Figures S5). In the NOE assignment process, difficulties were
6
136 | Org. Biomol. Chem., 2011, 9, 6133–6142
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View Article Online
encountered due to the duplication of the Ala-Arg-Lys sequence at
–9 and 11–12 positions. Nevertheless, the two distinct triads could
It should be emphasized that these remote contacts were only
7
found for glycopeptide 1 but not for the parent peptide or for the
1
0
be differentiated by assigning key NOEs for contacts between
the 7–9 motif and the N-terminal region, as well as between
the C-terminal part and the 11–12 repeated sequence. The rather
small values for most JNH-CHa coupling constants, together with the
detection of typical a-helix NOE patterns, was initially interpreted
as proof of the presence of a certain proportion of helical structures
in N/OFQ and the glycopeptide micellar samples. These findings
are in agreement with those reported by Mayo and co-workers
and with our above-mentioned CD data in TFE for the natural
other analogues, substituted at Ser by either b-GlcNAc or a-
GalNAc. These molecules do not show any secondary structural
features other than those related to the presence of a certain
percentage of a-helix.
Glycopeptide modeling
It is well known that O-glycosylation of Ser and Thr has a profound
5
0
effect on the conformation of the underlying peptide backbone,
however, such effect may be context dependent.
51,52
30
This effect also
analogue. These a-helix-type NOE contacts were observed for
all the peptide chains, including the FGGF message region at
the N-terminus (see S.I. Figures S6–S9). This is the case, for
regulates the 3D-orientation of the sugar attached to Ser or Thr,
and therefore, the study of the bioactive conformation of a given
glycopeptide obviously requires the careful analysis of both the
conformation of the peptide backbone and the orientation of the
sugar. Thus, 3D models of the glycopeptides were built employing
the experimental NMR data by using a standard protocol of
combined automated NOE assignment and structure calculations
1
6
13
16
13
instance, in the NOEs Hb2 Asn -Hd Lys , Hb3 Asn -Ha Lys ,
1
4
17
17
17
12
Ha Leu -Hb Gln and Hg Gln , Hd Leu14-Hg Gln , Hg Arg -
9
9
12
Ha Lys and Hg Lys -Hd Arg, which correspond to the N/OFQ
parent peptide (see S.I. Figure S10). However, some differences
2
in the NOE patterns could be detected at Gly in all the different
53
7
employing the CYANA program (see S.I., Table S9, for the
statistics of the NMR solution structures of the N/OFQ peptide
and glycopeptides 1–3).
analogues and at the Ala position of glycopeptide 1 (see below).
Conformation–activity relationship
The 3D model for N/OFQ and glycopeptide 2
The initial perspective provided by the above reported NOE
patterns suggests that helical structures are actually present for
all these molecules. We also estimated the chemical shift indexes
Fig. 4 shows the estimated Ha CSI values and the calculated 3-
D model for the parent peptide (N/OFQ) and glycopeptide 2.
Inspection of the Ha CSI values for both peptides indicates that
several residues from Ala7 to Gln17 seem to be integrated within
a helical structure. Indeed, as discussed above, the analysis of
(
CSI), which measure the deviation from random coil values
of the different backbone a protons (Ha). As reference values
to calculate the Ha CSIs, the literature values reported for the
random coil chemical shifts of the different amino acids were
48
used. As reference values for the CSIs of the glycosylated
positions, the reported values for non-glycosylated Ser and Thr
residues in random coil were employed in a first approximation.
In all cases, the data were in agreement with the presence of certain
populations of a-helix structures, especially at the C-terminus, but
with different lengths depending on the particular peptide.
However, in principle, this ubiquitous presence of a-helix does
not explain the differences in affinity for the NOP receptor
5
observed among the studied analogues. The Thr analogue
(
glycopeptide 1) is the most notorious compound of this series,
showing an affinity value of more than one order of magnitude
smaller than that measured for the parent compound. In this
context, the NOESY spectrum of glycopeptide 1 in SDS micelles
revealed some key contacts not detected in the spectra of the other
three peptides. Indeed, remote contacts between residues 4, 5, and
7
with the C-terminus 17 amino acid could be clearly identified.
In particular, non ambiguous NOEs between He21 Gln17 and
NH, Ha, Hb and Hg of Thr5, He21 Gln17-NH Ala 7 and He
Gln17-Hb2 Phe4 were detected, as shown in S.I. Figure S11. These
data can only be explained by the presence of folded structures for
glycopeptide1, whichcouldnotbe detected for the other analogues
(
see below). The presence of a folded structure can be tentatively
5
related with the presence of the a-GalNAc glycosylation at Thr .
Indeed, folded structures seem to be an intrinsic structural feature
49
of Thr residues when they are glycosylated by a-GalNAc.
Interestingly, glycopeptide 1 still preserves the characteristic NOE
pattern of an a-helix structure at its C-terminus, suggesting that
the observed folding is compatible with a certain population of a
helix moiety between residues 14 and 17.
Fig. 4 Superimposition of 8 selected structures, NOE connectivities
and Ha chemical shift index (CSI) for nociceptin (N/OFQ) and the
Ser -O-a-D-GalNAc glycopeptide 2.
10
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1
0
◦
◦
the NOE connectivities indicated that Ser -O-a-D-GalNAc has
additional i/i + 4 NOE contacts. Accordingly, it seems that a-
Thus, considering f
g
= 60 and y
g
= 180 , only the combination of
several c1 values could explain the experimental NOE contacts.
For instance, the conformation with c1 = -60 may explain the
H8–NH Ser10 NOE, while c1 = 60 could account for the H4–
NH Ser10 NOE. In turn, c1 = 180 is related to the H4–Hb Ala 7
NOE. Therefore, O-Ser-b-D-GlcNAc glycosylation of nociceptin
presents a high flexibility at the glycosylation site, somewhat
extended along the peptide chain.
1
0
◦
GalNAc glycosylation of Ser does not affect the helical structure
of the parent nociceptin. Furthermore, the analysis of the f/y
values of the 20 structures obtained by CYANA with respect to
the peptide backbone of the Lys-Ser-Ala triad showed that the
spatial disposition of these three residues is in good agreement
with a major a-helix structure. (See S.I. in Figures S12 and S13 for
the Ramachandran plots obtained by CYANA for these peptides).
In the case of glycopeptide 2, further conformational analysis of
the glycosidic torsion angles provided evidence that, in accordance
◦
◦
The 3D model for glycopeptide 1
with the exo-anomeric effect, f
g
(O5–C1–O1–Cb) was restricted
The initial analysis of the NMR data discussed above supports
that an a-O-GalNAc glycosylation at the Thr residue of O/NFQ
◦
5
at around 60 , while y
g
(C1–O1–Cb–Ca), besides being more
flexible, displayed a major anti-arrangement for the GalNAc
significantly affects the conformation of the peptide backbone.
48
5
residue. In contrast, c1 (N2–Ca–Cb–O1) seems to be much
The unique experimental remote contacts observed for (Thr -O-
more flexible. In fact, two different orientations around this torsion
a-D-GalNAc) (see S.I. Figure S11) could only be explained by the
presence of a folded conformation of glycopeptide 1 (Fig. 6 and
in S.I. Figure S18). The structure calculations revealed a cluster
of 8 folded hairpin-like conformers from the 20 best CYANA
structures. The analysis of f/y values (peptide backbone) of
◦
◦
angle (c1 = 60 and c1 = -150 ) were observed in solution (Fig. 5),
which were experimentally supported by the exclusive NOE cross-
peaks assigned for each conformation in the NOESY spectra (see
S.I., Figure S14 and S15).
5
Thr also located key changes in the secondary structure of this
molecule. For glycopeptide 1, the f/y values are concentrated
in the region of ppII in the Ramachandran plot, while for the
parent O/NFQ peptide, the torsion angles are placed in the a-
helix region. These confined values of f
g
, y and also of c1 reduce
g
the flexibility and consequently drive the peptide chain to adopt
folded conformations (Fig. 6). This effect is also in agreement
with the fact that rotation around the glycosidic linkage in
Fig. 5 Superimposition of the 3D view of the model structures for
10
◦
◦
glycopeptide 2 (Ser -O-a-D-GalNAc) with c1 = 60 and c1 = -150 . The
two distinct orientations of the GalNAc moiety are evident.
The 3D model for glycopeptide 3
As already discussed, the initial analysis of the NMR data
1
0
for glycopeptide 3, showing a Ser -O-b-D-GlcNAc modification,
revealed that more than one structural motif was again present
in solution. Indeed, the 3D model shown in Figure S16 shows
that, in spite of the presence of a helical structure at the C-
terminus, the flexibility of the glycopeptide remains very high.
Such an increase of flexibility after glycosylation is reflected by
the values of f and mainly y dihedral angles of the peptide
backbone (see S.I. Figure S17). The analysis of these torsion
angles with respect to the Lys-Ser-Ala triad also allowed us to
verify that, in contrast to the observations for peptide N/OFQ and
glycopeptide 2, several conformers have a wide range of torsion
angle values. This observation is in agreement with recent studies
that have demonstrated that b-O-GlcNAc glycosylation on a Ser
moiety introduces a higher flexibility around the glycosidic torsion
Fig. 6 Top left: Superimposition of 8 selected structures of the backbone
of glycopeptide 1 (Thr -O-a-D-GalNAc) according to the CYANA calcula-
tions, showing the folded structure. Top right panel: the NOE connectivities
and the Ha chemical shift index (CSI) are shown. The orientation of the
GalNAc residue versus the polypeptide chain is also shown at the bottom.
angles f
g
, y
g
4
and c1 of the lateral chain if compared to Thr-b-
5
5
O-GlcNAc. In this case, two possible values of f
g
, correlated
with minor fluctuations around y, were found. As expected, a
remarkable flexibility around c1 was observed for glycopeptide 3.
6
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Thr-a-D-GalNAc moieties is restricted, a feature that does not
take place in the Ser glycosylated analogues.
abiochem AG. Other reagents including tri-O-benzyl-D-galactal
and sodium dodecyl sulfate-d25 were purchased from Sigma-
Aldrich (St Louis, MO). [ H]-N/OFQ (82.3 Ci mmol ) was
obtained from Perkin-Elmer (Boston, MA). All other reagents
used were of analytical grade.
55
3
-1
Conclusions
The effect of O-glycosylation on the biological activity and
secondary structure of native nociceptin has been examined
for the first time by conjugation with GlcNAc and GalNAc
monosaccharides. The pharmacological analysis of the three new
glycopeptides revealed that N/OFQ glycosylation modulates the
binding affinity of this endogenous ligand to the drNOP receptor.
These effects were dependent on the glycosylation site as well as
the nature of the linked monosaccharide.
Comparative structural analysis between the glycopeptides and
the parent N/OFQ peptide by circular dichroism and NMR tech-
niques indicated that in water solutions random conformations
were mainly present. As seen by circular dichroism, upon addition
of TFE, a-helix structures started to be populated for all peptides.
In pure TFE solutions, the a-helix proportions were estimated
to be around 25% except for glycopeptide 3, which was only
Peptide synthesis
Nociceptin peptide (N/OFQ) and the glycopeptides (1, 2, and
a
3
) were synthesized manually by using standard N -Fmoc solid-
phase methodology on a FmocGln(Trt) prederivatized Wang
56
resin. The required glycosyl amino acid building blocks were pre-
31–33
a
pared using previously published methods.
N -Fmoc glycosyl
amino acids (3 equiv.) were used. Side chain protecting groups
used to build the glycopeptide sequences were the following: Trt
for Gln and Asn, tBu for Ser and Thr, Boc for Lys and Pmc
for Arg. The FmocGln(Trt) Wang resin (1 equiv.) was placed in
a glass peptide synthesis column with a frit on the bottom and
swollen in DMF for 1 h. The amide couplings were effected by
DIC (3 equiv.) and HOBt (6 equiv.). In the case of glycosylated
building blocks (3 equiv.) couplings were effected using PyBOP (3
equiv.), HOBt (3 equiv.) and DIEA (6 equiv.). Each coupling was
performed manually in this peptide synthesis column using DMF
as a solvent under reciprocal oscillating agitation. The coupling
efficiencies were monitored by the Kaiser ninhydrin test. The Fmoc
groups were removed with a solution of 20% piperidine in DMF
1
5%. Under these experimental conditions, the differences in the
proportion of a-helix do not correlate with the observed binding
affinities of these compounds and do not account for the large
decrease shown by glycopeptide 1.
In good agreement with previous reports, our NMR studies have
identified a certain degree of helical structure in the parent N/OFQ
peptide micellar solutions of SDS. The observed NOE patterns
for the glycopeptides suggest that a-helix motifs are also present
in the membrane mimic media, although glycopeptide 3 seems
to be more flexible than 2 and the parent compound. Fittingly,
for glycopeptide 1, a distinct set of remote contacts between
the N- and C-termini were detected in the NOESY spectra,
suggesting that this glycopeptide exhibits a significant population
of folded hairpin-like structures. The experimental NMR data
were employed to derive 3D models of these molecules, which
showed the presence of differently extended a-helix sections for
all of them, but also the presence of a unique folded conformation
only for glycopeptide 1. This distinct structural feature may be
responsible for the observed reduced affinity of this molecule for
the NOP receptor (nearly one order of magnitude). The difference
in binding affinity for the other analogues could be related to their
varying flexibility.
(
1 ¥ 9 min). The deprotected resin was washed with DMF, DCM,
and then DMF. After assembling the glycopeptide sequence, a
cocktail of TFA/TIS/H O (95 : 2.5 : 2.5) was used to remove the
2
side chain protecting groups and to cleave the peptide from the
resin. The crude glycopeptides were precipitated with ice-cold tert-
butyl methyl ether, filtered, redissolved in water and lyophilized.
The acetyl protecting groups on the sugar moieties were next
hydrolyzed carefully adding sodium methoxide/MeOH solutions
to methanol solutions of the protected glycopeptides (monitored
by analytical reverse-phase HPLC). The glycopeptides were neu-
tralized by addition of AcOH and concentrated under reduced
pressure. The final glycopeptides were purified by RP-HPLC using
acetonitrile–water gradient followed by lyophilization. The final
pure peptides were characterized by HRMS.
Finally, although on many occasions the relationships between
structure and activity seem elusive, in this case it is tempting to
justify that nociceptin analogues showing linear helical structures
are more complementary and thus interact more efficiently with
the native NOP receptor than folded structures. This hypothesis
is initially supported by the evidence provided here for the
Circular dichroism
CD spectra were obtained on a JASCO J-600 spectropolarimeter
and recorded in the range of 270–185 nm by using a 1 mm
cylindrical quartz cell. All spectra were recorded at room tem-
perature, with a band width of 1.0 nm, and a step resolution
of 0.2 nm. A sensitivity of 20 mdeg, a time constant of 2 s,
a scan speed of 20 nm min , and four scans were used. Prior
to measurements, the spectropolarimeter was calibrated with
a standard solution of ammonium d-camphor-10-sulfonate in
distilled water. The molar ellipticities were determined using the
formula [q]n→p* = qobsMRW/[10lc], where the qobs. is in millidegrees,
MRW is the mean residue molecular weight, l is the cell path
1
0
different behaviour of N/OFQ and its Ser glycosylated analogues
-
1
5
compared with those of Thr . In any event, structural studies
of the binding of these molecules to NOP receptor preparations
should shed further light on the interaction mechanisms and on the
bioactive conformation of nociceptin. These studies are currently
being performed in our laboratories.
-
1
Experimental
length in cm, and c is the glycopeptide concentration in mg mL .
The a-helicity percentage (f ) was determined by the Lifson–Roig
Materials
57
45
helix–coil theory and using the formula of Baldwin % helix =
[q]n→p*/[(-44 000 + 250T)(1 - 3/n)]100, where n represents the
number of amide bonds (including C-terminal amide) in the
All amino acid building blocks, coupling reagents and pred-
erivatized Fmoc Gln(Trt) Wang resin were purchased from Nov-
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glycopeptide and [q]n→p* is molar ellipticity of n→p* transition
and Ramachandran plots. Root mean square deviation (rmsd)
values were calculated using CYANA for superimpositions of the
backbone N, Ca and C¢ atoms, that is, the heavy atoms over the
whole peptide. To obtain the rmsd of a structure represented by
a bundle of conformers, all conformers were superimposed upon
the averaged one and the average of the rmsd values between
the individual conformers and their average coordinates was
calculated.
band at 222 nm.
NMR Spectroscopy
Experiments were recorded in H
2
O/D O 90 : 10 (phosphate buffer
2
PBS pH = 6.7) on Bruker Avance 600 and 800 MHz spectrometers
equipped with a triple channel cryoprobe and 278 K. NMR
assignments were accomplished using standard 2D-TOCSY (mix-
ing times of 20, 60, 80, and 100 ms), assisted by 2D-NOESY
experiments (mixing times of 200 and 300 ms). The concentration
of nociceptin and its glycopeptides for the NMR experiments
varied from 1 to 2 mM. The pH for the nociceptin sample was
Membrane preparation
Stably transfected HEK293 cells expressing the zebrafish NOP
receptor (drNOP) were grown in DMEM (GibCo BRL, UK)
medium supplemented with 10% fetal calf serum (GibC BRL,
1
0
adjusted to 5.4, and set at 6.7 for [Ser -O-b-D-GlcNAc]-N/OFQ,
1
0
5
[
Ser -O-a-D-GalNAc]-N/OFQ, [Thr -O-a-D-GalNAc]-N/OFQ
-
1
-1
to properly monitor the amide hydrogens of the polypeptide
backbone.
Additional NMR experiments were performed in the presence
of SDS micelles to mimic a membrane-like environment. In this
UK), 2 mM glutamine, 100 U mL penicillin, 0.1 mg mL
streptomycin (BioWhitakker, Walkersville, MD) and 0.25 mg
-
1
◦
mL Geneticin (GibCo BRL, UK) at 37 C under a 5% CO
2
atmosphere. Cells were grown to 80% confluence, harvested in PBS
pH 7.4 containing 2 mM EDTA and collected by centrifugation
case, the experiments were recorded in H
2
O/D O (90 : 10) on
2
◦
the same spectrometers and at 288 K. The NMR assignments
were accomplished using the same experiments mentioned above.
The concentration of nociceptin and its glycopeptides for the
NMR experiments again varied from 1 to 2 mM, while the
SDS concentration was 132 mM. The resonance of 2,2,3,3-
tetradeutero-3-trimethylsilylpropionic acid (TSP) was used as a
at 500g. The cell pellets were frozen at -80 C for at least 1 h
and resuspended in 50 mM Tris HCl buffer pH 7.4 (assay buffer)
-
1
with protease inhibitors (0.1 mg mL bacitracin, 3.3 mM captopril
and protease inhibitor cocktail, from Sigma-Aldrich, St Louis,
MO). The cell suspensions were homogenized with a Potter-
Elvehjem tissue grinder with a Teflon pestle in assay buffer, the
1
◦
chemical shift reference in the H NMR experiments (dTSP = 0
homogenates were centrifuged at 1500 rpm for 10 min at 4 C. The
ppm).
nuclear pellet was homogenized again, centrifuged and discarded.
The two supernatants were combined, homogenized again with
the tissue grinder and the membrane pellet was collected upon
Structure determination
◦
centrifugation at 18000g for 30 min at 4 C. The crude membrane
Peak lists for the NOESY spectra (T = 288 K, 300 ms) recorded
fraction was resuspended in ice-cold assay buffer with protease
inhibitors and protein concentration was determined by Bradford
(BioRad).
in presence of SDS micelles were generated by interactive peak
58
picking using the CARA software. NOESY cross-peak volumes
were determined by the automated peak integration routine. The
three-dimensional structure of nociceptin and its glycosylated
derivatives was determined using the standard protocol of com-
bined automated NOE (nuclear Overhauser effect) assignment
and the structure calculation of the CYANA program (version
Competition binding assays
The following unlabelled ligands were used: nociceptin (N/OFQ),
53
5
2
.1). Seven cycles of combined automated NOESY assignment
the glycosylated nociceptin analogues: glycopeptide 1 [Thr -
1
0
and structure calculations were followed by a final structure cal-
culation. The structure calculation started in each cycle from 100
randomized conformers, and the standard simulated annealing
O-a-D-GalNAc]-N/OFQ, glycopeptide 2 [Ser -O-a-D-GalNAc]-
10
N/OFQ, and glycopeptide 3 [Ser -O-b-D-GlcNAc]-N/OFQ. 20–
25 mg protein were incubated with different concentrations of
unlabelled ligand ranging from 0.3 nM to 10 mM, and using
59
schedule was used. The 20 conformers with the lowest final
CYANA target function values were retained for analysis and
passed to the next cycle. Weak restraints on glycosidic f/y
torsion-angle pairs and on side-chain torsion angles between
tetrahedral carbon atoms were applied temporarily during the
high-temperature and cooling phases of the simulated annealing
schedule in order to favour the permitted regions of the Ra-
machandran plot and staggered rotamer positions respectively.
The list of upper-distance bonds for the final structural calculation
consists of unambiguously assigned upper-distance bonds and
does not require the possible swapping of diastereotopic pairs. The
resulting 20 CYANA conformers represent the ensemble average
solution structure of nociceptin and analogues.
3
[ H]-N/OFQ as a radioligand (the working concentration was
◦
similar to the K
D
). Reactions were incubated for 1 h at 25
C
-
1
in a final volume of 250 mL of assay buffer with (0.1 mg mL )
proteinase-free BSA to avoid the adsorption of the radioligand to
the walls of the tubes. 10 mM nociceptin was used to determine
nonspecific binding. After incubation, the reaction was stopped
by adding 4 mL of ice-cold 50 mM Tris HCl buffer pH 7.4.
The mixture was rapidly filtrated using a Brandel Cell Harvester
and washed two times onto GF/B glass-fiber filters that were
presoaked with 0.2% polyethylenimine for at least 1 h. The filters
were placed in scintillation vials and incubated overnight at room
temperature in EcoScint A scintillation liquid (London, England).
Radioactivity was counted using a Beckman Coulter scintillation
counter (Pasadena, CA). All experiments were performed in
triplicate and repeated three times.
60
The MOLMOL program was used to visualize the three-
dimensional structures obtained by CYANA. CYANA was used
to obtain statistics on target function values, restraint violations
6
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Data analysis
18 R. Guerrini, G. Calo, A. Rizzi, C. Bianchi, L. H. Lazarus, S. Salvadori,
P. A. Temussi and D. Regoli, J. Med. Chem., 1997, 40, 1789–
1793.
Specific binding was defined as the difference between total
binding and nonspecific binding, as measured in presence of 10 mM
unlabelled nociceptin. Data were analyzed using the Graph Pad
1
9 R. Guerrini, G. Calo, A. Rizzi, R. Bigoni, C. Bianchi, S. Salvadori and
D. Regoli, Br. J. Pharmacol., 1998, 123, 163–165.
2
0 R. Guerrini, G. Calo, D. G. Lambert, G. Carr a´ , M. Arduin, T.
A. Barnes, J. McDonald, D. Rizzi, C. Trapella, E. Marzola, D. J.
Rowbotham, D. Regoli and S. Salvadori, J. Med. Chem., 2005, 48,
Prism software (San Diego, CA) and inhibition constants (K -
i
values) were calculated using the Cheng and Prusoff equation,
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1
421–1427.
2
2
2
2
2
1 M. Arduin, B. Spagnolo, G. Calo, R. Guerrini, G. Carr a` , C. Fischetti,
C. Trapella, E. Marzola, J. McDonald, D. G. Lambert, D. Regoli and
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61
site (K ). In all cases, data were fitted to the one-site or two-site
D
2 G. Calo, R. Guerrini, R. Bigoni, A. Rizzi, G. Marzola, H. Okawa, C.
Bianchi, D. G. Lambert, S. Salvadori and D. Regoli, Br. J. Pharmacol.,
competition model, and compared by using the nonlinear least-
squares curve-fitting which is based upon a statistical F-test.
2
000, 129, 1183–1193.
3 G. Calo, A. Rizzi, D. Rizzi, R. Bigoni, R. Guerrini, G. Marzola, M.
Marti, J. McDonald, M. Morari, D. G. Lambert, S. Salvadori and D.
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Marat o´ de TV3 (Pain, project reference 070430-31-32-33). We
thank F. J Ca n˜ ada, M. Morando and L. Nieto for helpful
discussions, and M. Bruix for the access to the 800 MHz
NMR spectrometer. JJB acknowledges Ministerio de Ciencia
e Innovaci o´ n (Spain) (CTQ2009-08536) for financial support.
F. M. thanks to FCT-Portugal for a post-doc research grant
9
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