Synthesis and biological activity of novel small peptides with aminophosphonates moiety as NOP receptor ligands

Article abstract of DOI:10.1007/s00726-010-0624-1

The aim of the present study was the synthesis and the biological screening of new analogs of Ac-RYYRWK-NH₂, modified at the N-terminal with 1-[(methoxyphosphono)methylamino]cycloalkanecarboxylic acids. The four newly synthesized ligands for the nociceptin/orphanin FQ (N/OFQ) receptor (NOP) have been prepared by solid-phase peptide synthesis-Fmoc-strategy. These compounds were tested for agonistic activity in vitro on electrically stimulated smooth-muscle preparations isolated from vas deferens of Wistar rats. Our data showed that substitution of Arg at position 1 with aminophosphonates moiety decreased significantly the affinity of ligands to the NOP receptor. Furthermore, the enlargement of the cycle (with 5-8 carbon atoms) additionally diminished both the activity and the selectivity for NOP-receptor.

Full text of DOI:10.1007/s00726-010-0624-1

Amino Acids (2010) 39:1537–1543
DOI 10.1007/s00726-010-0624-1
ORIGINAL ARTICLE
Synthesis and biological activity of novel small peptides
with aminophosphonates moiety as NOP receptor ligands
•
Emilia D. Naydenova Petar T. Todorov
•
•
Polina I. Mateeva Rositza N. Zamfirova
•
•
Nikola D. Pavlov Simeon B. Todorov
Received: 19 March 2010 / Accepted: 7 May 2010 / Published online: 25 May 2010
Ó Springer-Verlag 2010
Abstract The aim of the present study was the synthesis
and the biological screening of new analogs of Ac-RY-
YRWK-NH2, modified at the N-terminal with 1-[(meth-
oxyphosphono)methylamino]cycloalkanecarboxylic acids.
The four newly synthesized ligands for the nociceptin/
orphanin FQ (N/OFQ) receptor (NOP) have been prepared
by solid-phase peptide synthesis—Fmoc-strategy. These
compounds were tested for agonistic activity in vitro on
electrically stimulated smooth-muscle preparations isolated
from vas deferens of Wistar rats. Our data showed that
substitution of Arg at position 1 with aminophosphonates
moiety decreased significantly the affinity of ligands to the
NOP receptor. Furthermore, the enlargement of the cycle
(with 5–8 carbon atoms) additionally diminished both the
activity and the selectivity for NOP-receptor.
and Pasternak 2001; Dzhambazova et al. 2008) by selec-
tively activating an opioid-like receptor named ORL1 or
N/OFQ peptide (NOP) receptor (Cox et al. 2000), a novel
member of the opioid receptor family.
By selective activation of G-protein coupled receptor
(NOP), N/OFQ produces inhibition of adenylate cyclase
(Meunier et al. 1995; Reinscheid et al. 1995), activation of
inward K? current (Matthes et al. 1996), and inhibition of
Ca2? channels (Connor et al. 1996). Moreover, recent data
indicate that NOP antagonists are worthy of developing as
innovative antiparkinson drugs (Marti et al. 2005). Hence,
the NOP receptor system is highly attractive molecular
target for drug development purposes (Zaveri 2003). The
understanding of the role of the N/OFQ/NOP system
depends upon the development of selective and highly
potent peptide and non-peptide agonist and antagonist
ligands. Among them are the N/OFQ related peptides
(Guerrini et al. 2000; Naydenova et al. 2006; Arduin et al.
2007) and small peptides, identified by screening of peptide
combinatorial libraries (Dooley et al. 1997).
Keywords Nociceptin/orphanin FQ ꢀ NOP receptor ꢀ
Nociceptin analogs ꢀ SPPS ꢀ Aminophosphonates ꢀ
Rat vas deferens
Arduin et al. reported the synthesis and biological
evaluation of a series of N/OFQ-NH2 analogs substituted in
position 7 and 11 with Ca,a,-disubstituted cyclic, linear,
and branched amino acids compatible with alpha helix
structures (Arduin et al. 2007). The most interesting results
in terms of biological activity have been obtained using
Aib in position 7. This substitution was then combined with
other chemical modifications and series of novel highly
potent peptide ligands for the NOP receptor were gener-
ated. This receptor is also activated by non-peptide agonists
(Jenck et al. 2000). Recently, a potent and selective non-
peptide antagonist has been reported (Noda et al. 1998;
Ozaki et al. 2000), but it has been difficult to identify any
general structural elements, common to all of these organic
compounds.
Introduction
Nociceptin/Orphanin FQ (N/OFQ) is a heptadecapeptide
(Meunier et al. 1995; Reinscheid et al. 1995), which
modulates a number of biological functions in the central
and the peripheral nervous system (Calo et al. 2000; Mogil
E. D. Naydenova (&) ꢀ P. T. Todorov ꢀ N. D. Pavlov
Department of Organic Chemistry, University of Chemical
Technology and Metallurgy, 1756 Sofia, Bulgaria
e-mail: e_naydenova@abv.bg
P. I. Mateeva ꢀ R. N. Zamfirova ꢀ S. B. Todorov
Institute of Neurobiology, Bulgarian Academy of Sciences,
1113 Sofia, Bulgaria
123

1538
E. D. Naydenova et al.
Dooley et al. screened a synthetic peptide combinatorial
other reagents and solvents were analytical or HPLC grade
and were bought from Merck (Germany).
library, and isolated and characterized several hexapeptides,
including Ac-RYYRWR-NH2, Ac-RYYRWK-NH2 and
Ac-RYYRIK-NH2, which have high affinity for the NOP
receptor. These hexapeptides are not only significantly
smaller than N/OFQ-[1-13]-NH2, the shortest N/OFQ
fragment with the same affinity and biological potency as
the parent peptide, but they also exhibit distinctive phar-
macological profiles (Calo et al. 2000). The hexapeptide
Ac-RYYRWK-NH2 has been described as a potent partial
agonist of the NOP receptor, without affinity to l-, j- or
d- opioid receptors (Dooley et al. 1997). In vivo, Ac-
RYYRWK-NH2 has been shown to increase food intake in
rats similarly to N/OFQ, and to mimic N/OFQ in mouse
colon preparation (Mason et al. 1998; Rizzi et al. 1999).
In particular, the hexapeptide Ac-RYYRIK-NH2 has been
shown to competitively inhibit nociceptin (NC)-induced
guanosine-50-O-(3-thio)trisphosphate-c-S (GTPcS) binding
to the NOP receptor (Berger et al. 1999, 2000). These
hexapeptides contain three positively charged basic residues
similar to N/OFQ, which has four positively charged amino
acids at the C-terminus. In an Ala-scanning study, two Arg
residues, at positions 1 and 4, were found to be important for
binding to the NOP receptor (Kawano et al. 2002).
The solid-phase peptide synthesis by Fmoc (9-fluor-
enylmethoxycarbonyl) chemistry was used to obtain noci-
ceptin/orphanin FQ ligands. Rink-amide resin was used as a
solid-phase carrier, and TBTU was used as a coupling
reagent. The 3-functional amino acids were embedded as
follows: Arg—as Fmoc-Arg(Pbf)-OH, Lys—as Fmoc-Lys
(Boc)-OH, Trp—as Fmoc-Trp(Boc)-OH, Tyr—as Fmoc-
Tyr(tBu)-OH. The coupling reactions were performed using
for amino acid/TBTU/HOBt/DIEA/resin a molar ratio of
3/2.9/3/6/1, in a 1:1 mixture of DMF/DCM. Piperidine in
dimethylformamide (20% solution) was used to remove the
Fmoc group at every step. The coupling and deprotection
reactions were checked by the Kaiser test. All the 1-[(di-
methoxyphosphono)methylamino] cycloalkanecarboxylic
acids (3 equiv) were coupled to the growing peptide chain by
using 2-(1H-Benzotriazole-1-yl)1,1,3,3-tetramethyluronium
tetrafluoroborate (TBTU, 3 equiv) in the presence of an
equimolar concentration of 1-hydroxy benzotriazole (HOBt),
dissolved in N,N-dimethylformamide (DMF) at excess of
N,N-diisopropylethylamine (DIEA), the coupling reaction
time being 15 h. The cleavage of the synthesized peptides
from the resin was done, using a mixture of 95% trifluoro-
acetic acid (TFA), 2.5% triisopropylsilane (TIS), and 2.5%
water. After filtration of the exhausted resin, the solvent was
concentrated in vacuum and the residue triturated with cool
ether. The peptides were analyzed on a reversed-phase high-
performance liquid chromatography (HPLC) C18 column:
flow 1 ml/min, H2O/CH3CN/0.1% TFA gradient 0 ? 100%
20 min. The peptide purity was checked by electrospray
ionization massspectrometry. The analytical data for the
synthesized peptides prepared were as follows: compound 3
tR 5.40 min, [98% pure, 1032.5 calculated (MH?), 1033.0
observed (MH?); compound 4 tR 5.60 min, [99% pure,
1060.5 calculated (MH?), 1060.8 observed (MH?); com-
pound 5 tR 5.80 min,[99% pure, 1074.5 calculated (MH?),
1075.0 observed (MH?); compound 6 tR 5.06 min, [99%
pure, 1203.6 calculated (MH?), 1203.1 observed (MH?).
Phosphonopeptides and phosphinopeptides are phospho-
rus analogs of naturally occurring peptides. Their impor-
tance is obvious from the fact that they have been widely
used as enzyme inhibitors and as happens for catalytic
antibody research because they can be considered as stable
mimetics of tetrahedral transition states in ester and amide
hydrolysis and formation (Kafarski and Lejczak 2000a, b;
Cunningham et al. 2008). Many methods are now available
for preparation of phosphinopeptides, containing C-terminal
a-aminoalkylphosphinic acids (Kukhar et al. 1994; Solos-
honok et al. 1992; Xu and Yu 1999; Meng and Xu 2010).
Presently, there are no data about synthesis and biological
activity of peptides containing N-terminal 1-[(methoxy-
phosphono)methylamino]cycloalkanecarboxylic acids.
Based on Ac-RYYRWK-NH2 as a chemical template,
we synthesized novel NOP-receptor short-chain analogs
modified at N-terminal with a-aminocycloalkane carbox-
ylic acid, bearing (methoxyphosphonyl)methyl group.
Besides, we investigated the structure–activity relationship
of the new analogs in vitro on electrically stimulated
smooth-muscle preparations from rat vas deferens.
Biological tests
The biological activity of the newly synthesized peptides
has been tested on electrically stimulated smooth-muscle
preparations. The dissected vasa deferentia were cleaned
from the surrounding tissues and blood vessels. Prostatic
segments (12–15 mm long) were fixed in 4 ml organ baths
and connected to electronic transducers. The preparations,
pre-loaded with 1 g, were left for a 60 min adaptation at
32.5°C in carbogen-aerated Krebs solution. Smooth-muscle
contractions were evoked by low-frequency electrical
stimulation with parameters: 0.05 Hz frequency, 1 ms
pulse duration, sub-maximal voltage.
Materials and methods
Synthesis
The protected amino acids and Fmoc-Rink Amide MBHA
Resin were purchased from IrisBiotech (Germany). All
123

Synthesis and biological activity of novel small peptides with aminophosphonates
1539
The smooth-muscle tone of the ES-induced contractions
was registered by on-line system. The tested compounds
In order to elucidate the influence of Arg and acetyl
group we decided to introduce the 1-[(methoxyphospho-
no)methylamino]cyclopentanecarboxylic acid to the N-side
of Arg, compound 6. This newly synthesized heptapeptide
has the following sequence:
were applied cumulatively in concentrations 10-10
–
10-4 M. In part of the experiments naloxone (Nal, 10-6M,
for to block all types opioid receptors, IUPHAR-DB) or
naloxone benzoylhydrazone (3 9 10-5M, as a blocker of
NOP receptors, Chiou 2001) was administered to the organ
bath 10 or 15 min prior to the peptides under investigation.
All compounds were dissolved in saline prior the
experiment. Ac-RYYRWK-NH2 was dissolved in 0.01 M
acetic acid.
O
HO
P
CH2 NH
CO
NH2
Arg Tyr Tyr Arg Trp Lys
H3CO
The crude peptides were purified on a reversed-phase
high-performance liquid chromatography (HPLC) and the
molecular weights determined using electrospray ioniza-
tion mass-spectrometry. The analytical dates are shown in
‘‘Materials and methods’’.
Animals
In the experiments, male Wistar rats (180–200 g), housed
at 22–25°C, were used. The animals were allowed an
adaptation period, with free access to food and water and a
natural day/night light cycle. All animals were killed under
light ether anesthesia.
Biological activity
All newly synthesized compounds induce a strong tachy-
phylaxis (similarly to Ac-RYYRWK-NH2; Dooley et al.
1997). That is why each cumulative dose–response curve
for the peptides (without or with blockade of opioid
and nociceptin receptors) was created on a separate vas-
deferens preparation.
The experiments were performed according to the
‘‘Principles of laboratory animals care’’ (NIH publication
No 85–23, revised 1985) and the rules of the ethic Com-
mittee of the Institute of Neurobiology, BAS.
The effects of newly synthesized short-chain peptides
(3–6) were compared with Ac-RYYRWK-NH2 (1), which
is known to be a very potent and selective partial agonist of
NOP receptors (Dooley et al. 1997). Therefore, it has been
synthesized in our laboratory as a referent compound.
Results and discussion
Chemistry
The peptides shown in Table 1 were prepared with good
yield by solid phase synthesis using TBTU, an efficient
peptide coupling reagent. The 1-[(dimethoxyphosphono)
methylamino]cycloalkanecarboxylic acids were previously
prepared by our group following Kabachnik- Fields reac-
tion (Naydenova et al. 2008, 2010). In view of the low
solubility of a-aminophosphonates, the coupling reaction
was performed in N,N-dimethylformamide (DMF) at high
excess of N,N-diisopropylethylamine (DIEA) and the
coupling reaction time was 15 h. During the TFA cleavage
one of the methoxy groups from aminophosphonic residue
was removed. In compounds 3–5, Arg at position 1 was
substituted by 1-[(methoxyphosphono) methylamino]
cycloalkanecarboxylic acids.
Tested on rat vas deferens, data (pEC50 = 8.9; Emax =
-40%) similar to those reported in the literature
(pEC50 = 9.1; Emax = -66%; Ho et al. 2000) were
obtained.
The removal of the acetyl group in the parent peptide (2)
dramatically reduced both the potency (pEC50 = 4.84) and
efficacy (Emax = -25%, Fig. 1). Furthermore, the
replacement of the acetyl group by aminophosphonates
moiety (compound 6, Fig. 2) also reduced the potency of
the peptide (pEC50 = 5.08). Nevertheless, its action
remains specific for NOP receptors: applied after opioid
receptor blockade by naloxone, the peptide does not change
its effect. The blockade of NOP receptors with naloxone
benzoylhydrazone prevented completely the inhibitory
effect of compound 6.
The new hexapeptides have the following sequences:
In the three hexapeptide derivatives (3, 4, and 5), Arg1
was substituted by 1-[(methoxyphosphono)methylamino]
cycloalkanecarboxylic acids with 5-, 7-, and 8-membered
rings. The enlargement of the cycle (compounds 4 and 5)
significantly diminished pEC50 and Emax. Practically, these
two compounds affect the smooth-muscle contractions only
in the highest concentration used (10-4M, Fig. 3), most
probably not by interactions with NOP receptors. In rela-
tively low concentrations (10-8–10-7M), compound 3
X Tyr Tyr Arg Trp Lys
NH2
O
C
O
P
HO
CH2 NH
H3CO
where X is :
(CH2)n
n = 0, 2, 3
123

1540
E. D. Naydenova et al.
Table 1 Effects and potency of the newly synthesized peptides on LFES-evoked contractions of rat vas deferens
Peptide tested
Peptide alone
Peptide ? Nal
Peptide ? Nal-B
pEC50
8.95
Emax
pEC50
9.37
Emax
-44.7
pEC50
6.8
Emax
-40.0
-25.0
-16.2
-13.9
RYYRWK NH2
(1)
Ac
H
RYYRWK NH2
(2)
4.84
5.82
4.94
4.83
-7.7
4.92
5.00
-3.5
O
-7.67
-4.75
HO
P
H3CO
NH2
CH2 NH
CO
YYRWK
(3)
O
HO
4.81
-7.4
4.86
-9.44
4.52
1.32
CH2 NH
P
CO
YYRWK NH2
H3CO
(4)
O
4.78
-3.25
4.85
-5.63
4.71
-0.4
HO
CH2 NH
P
CO
YYRWK NH2
H3CO
(5)
O
5.08
-25.5
5.19
-23.5
4.74
-2.6
HO
NH2
RYYRWK
CH2 NH
CO
P
H3CO
(6)
123

Synthesis and biological activity of novel small peptides with aminophosphonates
1541
modifications with other groups (pyvaloyl, formyl, ben-
zoyl, mesyl) in an investigation of Gu¨ndu¨z et al. (Gu¨ndu¨z
et al. 2006). Our results lead also to the conclusion that
when Arg1 is included in the hexapeptide molecule, the
affinity and selectivity for NOP receptor is preserved
(compound 6). It might be suggested that the smooth-
muscle inhibition by compounds 3–5 (where Arg1 is
replaced) is due to the interaction of these peptide analogs
with the classical opioid receptors and not with the NOP-
receptor. Dooley et al. established that the following
sequence Arg-Tyr-Tyr is the most effective at the first
three positions of NOP-receptor ligands (Dooley et al.
1997). Later on, structure–activity studies on hexapeptides
with modified amino acids confirm that Arg1 is preferred
and even ‘‘crucial’’ (Gu¨ndu¨z et al. 2006); moreover, it has
been shown that both Arg1 and Arg4 in the hexapeptide
chain are required for biological activity (Judd et al.
2004).
100
75
50
25
0
2
1
-11 -10 -9
-8
-7
-6
-5
log. conc.(M)
Fig. 1 Effects of Ac-RYYRWK-NH2 (1) and H-RYYRWK-NH2 (2)
on LFES-induced smooth-muscle contractions
100
75
It is known that the activation of all opioid receptors
requires a Tyr residue in position 1. There are many data
from structure–activity studies, showing that replacement
of Phe1 in nociceptin molecule with Tyr produces a pep-
tide, which activates NOP receptor (Butour et al. 1997;
Varani et al. 1998), as well as opioid l (Salvadori et al.
1997) and j (Reinscheid et al. 1998) receptors. However,
the shorter nociceptin analogs [Tyr1]NC(1–9)NH2 and
[Tyr1]NC(1–5)NH2 activate the classical opioid receptors
only (Varani et al. 1999).
50
25
6
1
0
-11 -10 -9
-8
-7
-6
-5
log.conc.(M)
Fig. 2 Effects of hexapeptide analogs (1 and 6) on LFES-induced
smooth-muscle contractions
Concerning compound 3, it should be stated that the
inhibitory effects of the compound after naloxone and
naloxone benzoylhydrazone are practically equal. This
suggests that compound 3 most probably is agonist of
opioid receptors only, since its activity is blocked by nal-
oxone, being not further affected by naloxone
benzoylhydrazone.
125
100
75
Our results suggest that the incorporation of 1-[(meth-
oxyphosphono)methylamino] cycloalkanecarboxylic acid
in position 1 of Ac-RYYRWK-NH2 decreases the N/OFQ-
like activity of the newly synthesized peptide analogs.
50
3
4
5
1
25
0
Acknowledgments This study was supported by Grant DTK_02/61
of the National Research Fund, Bulgaria.
-11 -10 -9
-8
-7
-6
-5
-4
log.conc.(M)
Fig. 3 Effects of Ac-RYYRWK-NH2 (1) and the hexapeptide
analogs (3–5) on LFES-induced smooth-muscle contractions
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