Kyotorphin analogues containing unnatural amino acids: Synthesis, analgesic activity and computer modeling of their interactions with μ-receptor

Article abstract of DOI:10.1007/s00044-014-0953-9

Kyotorphin (KTP; Tyr-Arg) an endogenous neuropeptide is potently analgesic when delivered directly to CNS. An effort to enhance the potency, enzymatic stability and improving bioavailability of KTP is the modification with unnatural amino acids. The aims of presented study were: (1) To synthesize new analogues of kyotorphin containing unnatural amino acids: norcanavaine (NCav) and norcanaline (NCan), structural analogues of arginine and ornithine, respectively; (2) To understand the influence of the arginine mimetics on the pharmacological properties of KTP analogues, through examination their effects on the paw pressure nociceptive threshold; (3) To find relationship between the structure and obtained biological effects of the all synthesized kyotorhin analogues, by molecular docking with μ-opioid receptor. As a result of our work four new kyotorphin analogues containing NCan and NCav were obtained. A correlation between the data from the in vivo test and docking results was found. This allows a better elucidation of the ligand-receptor interactions, the prediction of biological activity of the newly synthesized compounds, and to generate compounds with increased biological effects. Springer Science+Business Media 2014.

Full text of DOI:10.1007/s00044-014-0953-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-014-0953-9
ORIGINAL RESEARCH
Kyotorphin analogues containing unnatural amino acids:
synthesis, analgesic activity and computer modeling of their
interactions with l-receptor
•
Tatyana Dzimbova Adriana Bocheva
•
Tamara Pajpanova
Received: 30 July 2013 / Accepted: 11 February 2014
Ó Springer Science+Business Media New York 2014
Abstract Kyotorphin (KTP; Tyr-Arg) an endogenous
neuropeptide is potently analgesic when delivered directly
to CNS. An effort to enhance the potency, enzymatic sta-
bility and improving bioavailability of KTP is the modifi-
cation with unnatural amino acids. The aims of presented
study were: (1) To synthesize new analogues of kyotorphin
containing unnatural amino acids: norcanavaine (NCav)
and norcanaline (NCan), structural analogues of arginine
and ornithine, respectively; (2) To understand the influence
of the arginine mimetics on the pharmacological properties
of KTP analogues, through examination their effects on the
paw pressure nociceptive threshold; (3) To find relationship
between the structure and obtained biological effects of the
all synthesized kyotorhin analogues, by molecular docking
with l-opioid receptor. As a result of our work four new
kyotorphin analogues containing NCan and NCav were
obtained. A correlation between the data from the in vivo
test and docking results was found. This allows a better
elucidation of the ligand-receptor interactions, the predic-
tion of biological activity of the newly synthesized com-
pounds, and to generate compounds with increased
biological effects.
Abbreviations
KTP
CNS
MOR
PP
Kyotorphin
Central nervous system
l-Opioid receptors
Paw-pressure nociceptive test
Canavanine
Cav
NCav
Can
Norcanavanine
Canaline
NCan
Norcanaline
NsArg Norsulfoarginine
Piv-Cl Pivaloyl chloride
DIPEA N,N-diisopropylethylamine
Boc
Z
Di-tert-butyl dicarbonate
Carboxybenzyl
Introduction
Kyotorphin (KTP; L-Tyr-L-Arg) was first isolated from
bovine brain in 1979 (Takagi et al., 1979) and has subse-
quently been found in the brain of several mammals and in
human cerebrospinal fluid (Kolaeva et al., 2000).
The most studied characteristics of KTP are its structure,
biosynthesis and hydrolysis, the mechanisms of its release
from nerve terminals, its interaction with the postsynaptic
membrane and the binding to specific receptors (Kawabata
et al., 1996, 1993). Kyotorhin is an endogenous dipeptide
and may be formed by the biosynthesis from Tyr and Arg
(Ueda et al., 1982). It can be consider as a neurotrans-
mitter, satisfied all five criteria (Zakutskii et al., 2008): (1)
it is synthesized in synaptic endings; (2) it is stored in
synaptic vesicles (Ueda et al., 1982); (3) it is released at the
depolarization of presynaptic membranes (Ueda et al.,
1986a), (4) it is enzymatically inactivated by kyotorphin
hydrolase (Ueda et al., 1985), and (5) KTP interacts with
Keywords Arginine mimetics Á Kyotorphin Á
Nociception Á l-Opioid receptor Á Docking Á GOLD
T. Dzimbova (&) Á T. Pajpanova
Institute of Molecular Biology ‘‘Roumen Tsanev’’, Bulgarian
Academy of Sciences, Acad. G. Bonchev Str., Block 21,
1113 Sofia, Bulgaria
e-mail: tdzimbova@gmail.com; tania_dzimbova@abv.bg
A. Bocheva
Department of Pathophysiology, Faculty of Medicine, Medical
University of Sofia, 2, Zdrave Str, 1431 Sofia, Bulgaria
123

Med Chem Res
neutral pH and is involved in many important physiological
and pathophysiological processes. The guanidinium group
of arginine has a pKa = 12, has planar geometry and can be a
donor in the formation of up to five hydrogen bonds (Fig. 1).
The arginine has been found to play an important role in
binding of negatively charged compounds such as: sub-
strates; cofactors; and different effectors to the protein
active sites (Riordan et al., 1977). It is often involved in
formation of salt bridges, mainly with aspartate or gluta-
mate side chains. The arginine also plays numerous roles in
the cell metabolism (Fig. 2). Endogenously applied, L-Arg
acts as an effective precursor of kyotorphin and has an
antinociceptive effect in rats and mice (Kawabata et al.,
1993, 1996). Additionally, clinical studies demonstrated
naloxone-reversible analgesic effects of L-Arg in chronic
pain patients (Harima et al., 1991).
O
δ+
H
R
N
O
O
C
H
H
δ+
N
N
δ+
H
O
H
O
Fig. 1 Schematic presentation of guanidinium group hydrogen bond
formation with five different oxygen atoms
specific kyotorphin receptors on the postsynaptic mem-
brane. This leads to the activation of phospholipase C, and
an increase in the concentration of inositol-3-phosphate
(Ueda et al., 1985). These properties of KTP are typical for
the neurotransmitters, and it exerts nonopioid actions
independent of enkephalin release (Ueda and Inoue, 2000).
This fact point out the possibility of an indirect action of
KTP, which would not bind to the opioid receptors (l, d,
j), but it is involved in Met-enkephalin release (Takagi
et al., 1979). All this led to presumption that the dipeptide
bind to a specific receptor (kyotorphin receptor, KTPr)
(Ueda et al., 1986b), triggering a cascade of events that
could cause strong analgesia in the brain (Ueda et al., 1987,
Despite its interesting properties, KTP is only active
when injected directly into the brain. When systemically
administered, it only shows brief activity and at a high dose
of 200 mg kg-1 (Chen et al., 1998). This limited capacity
to cross the blood–brain barrier, mainly related to both
insufficient lipophilicity and susceptibility to various lytic
enzymes, confines KTP pharmacological applications. A
logical strategy to target KTP to the brain is the chemical
modification of KTP into analogues that are able to
maintain analgesic properties and are able to cross the
blood–brain barrier.
¨
2000). Despite several studies (Takagi et al., 1979; Bor-
jesson and Hu¨nenberger, 2001; Shiomi et al., 1981) con-
firmed the existence of kyotorphin receptor, it has not yet
been identified. There is still the question of whether the
We started to search for a KTP derivative with minimal
differences relative to KTP, focusing our attention on the
replacement of arginine with its analogues with reduced
basicity. Many efforts were made in the design and prep-
aration of arginine analogues with reduced basicity,
resulting in increased stability towards enzymatic degra-
¨
¨
kyotorphin receptor is specific (Borjesson and Hunenber-
ger, 2001), or it is the result of a mixed oligomerization of
l- and d-opioid receptors (George et al., 2000).
Even it is known that kyotorphin’s binding pocket has to
be different from the opioid receptors (Ueda et al., 2000;
Takagi et al., 1979; Hansen et al., 1992), there are obvious
similarities between KTP and most of the endogenous
opioid peptides and even with morphine (Machuqueiro and
Baptista, 2007). The L-Tyr residue at the first position of
the peptide is present in most of the opioid peptides, and is
believed to be crucial for receptor recognition due to both
p-stacking and hydrogen-bonding with the alcohol of the
phenol group. A protonable N-terminal group is also
present in KTP and when protonated, can form a salt bridge
with an anionic group in the receptor, which is typical for
the morphine/l-receptor interaction. Conformational stud-
ies of kyotorphin molecule (Machuqueiro and Baptista,
2007) suggest that, despite the fact that kyotorphin does not
bind to the opioid receptors, there is no indication for the
kyotorphin receptor pocket to be very different from those
of the opioid receptors.
ˇ ˇ
dation (Peterlin-Masic and Kikelj, 2001). In view of this, it
was interesting to design and synthesize unnatural amino
acids containing a guanidinium functionality (sulfo- and
oxyguanidinium), as structural analogue of arginine (Pa-
jpanova et al., 1997; Stanchev et al., 2000; Dzimbova
et al., 2003, 2007). The synthesis and evaluation of kyo-
torhins containing these arginine analogues was done.
Modification in the arginine residue of kyotorphin with
canavanine and sulfoarginine has also been shown to have
a significant influence on the analgesic activity (Bocheva
et al., 1996; Dzimbova et al., 2006).
In the present paper we describe a several of kyotorhins
that we designed with the goal to develop analogues with
increased degradation stability and potency. The design
was mainly based on structural modification of Arg resi-
due. Arg2 was replaced with unnatural amino acids (NCav
and NCan), structural analogues of canavanine and cana-
line, respectively. The reverse-kyotorphins were also pre-
pared. In order to understand the influence of the arginine
mimetics on the pharmacological properties of KTP
The L-Arg residue at the second position of kyotorphin
has guanidinium group, which is positively charged at
123

Med Chem Res
Fig. 2 Arginine metabolism
Amino acids and analogues
analogues, we studied their effects on the PP (paw-pres-
sure) nociceptive threshold. Next, assumed to find rela-
tionship between the structure and obtained biological
effects of the all synthesized kyotorhin analogues, molec-
ular docking with l-opioid receptor was applied. The
elucidation of ligand-receptor interaction would be helpful
for the discovery of new active analogs of KTP.
Amino acids D-Kyo, Kyo, L-Can, L-Cav, L-Cit, L-NAME,
and L-Orn were obtained from Sigma. The Cys(O2NH2)
was synthesized following original synthetic scheme pre-
sented by Aleksiev and Stoev (1971). The synthesis of
unnatural amino acids: L-NCan; NsArg; NsArg-NH2 and
NsArg-OBzl (Dzimbova et al., 2003, 2011) were previ-
ously reported.
Materials and methods
Peptide synthesis
Chemistry
The synthesis of the kyotorphin analogues: Tyr-Cav (Bo-
cheva et al., 1996), Tyr(Cl2)-Cav (Pajpanova et al., 1992)
and NsArg-Tyr, Tyr-NsArg, Tyr-NsArg-NH2, Tyr-NsArg-
OBzl (Dzimbova et al., 2006) were previously reported.
All chemicals were of analytical grade. All anhydrous
solvents were obtained commercially (Fluka) and used
directly. HPLC-grade acetonitrile and MeOH were pur-
chased from Merck. Analytical TLC was performed on
Merck silica gel (60F254) plates (0.25 mm) using of the
following solvent systems: (A) V (C6H6): V (CH3COCH3):
V (CH3COOH) = 100: 50: 2; (B) V (CHCl3): V (MeOH):
V (CH3COOH) = 95: 5: 5; C) V (CH3CN): V (H2O) = 4:
1. Visualization was done with either UV, ninhydrin or a
chlorine tolidine reagent. HPLC analyses were performed
on Agilent Technologies HP 1100 and Waters 2695 LC
Synthesis of Z-NCan-Tyr-OMe (methyl 2-(3-(aminooxy)-2-
(((benzyloxy)carbonyl)amino)propanamido)-3-(4-
hydroxyphenyl)propanoate) (23) and Z-NCav-Tyr-OMe
(methyl 2-(2-(((benzyloxy)carbonyl)amino)-3-
(guanidinooxy)propanamido)-3-(4-
hydroxyphenyl)propanoate) (24)
instruments, using
a
Column: LichrosphereÒ RP8
A solution of Z-NCan (Z-NCav) (0.5 mM) and NMM
(0.06 ml, 0.5 mM) in 5 ml DMF was cooled to -10 °C
and Piv-Cl (0.061 ml, 0.5 mM) was added dropwise. After
10 min a solution of HCl.Tyr-OMe (0.11 g, 0.5 mM) and
Et3N (0.07 ml, 0.5 mM) in 5 ml DMF was added. The
reaction mixture was stirred 4 h at room temperature.
Before it was quenched by the addition of water (10 ml)
and extracted with CHCl3 (3 9 10 ml). The combined
organic phases were washed consequently with 5 %
NaHCO3 (3 9 10 ml), 5 % NaHSO4 (3 9 10 ml), and
(100 9 4,6 mm); mobile phase: acetonitrile/deionized
1
water 40/60 (v/v). H NMR and 13C NMR spectra were
recorded on Bruker WM-250 and Avance DRX-250
(250 MHz and 75 MHz, respectively) spectrometers. Mass
spectra were recorded on a Fissons-Triple Quadrupol-ES
mass spectrometer. The IR-spectra were measured using a
Bomem-Michelson 100 FT-IR-spectrometer (4,000–400,
2 cm-1 resolution, 150 scans). Solid-state IR spectra were
recorded, using the KBr disk technique.
123

Med Chem Res
brine, dried over anhydrous Na2SO4, filtered and CHCl3 1H, CH), 3.44 (d, 2H, CH2), 2.0 (s, 2H, NH2), 1.38 (s, 9H,
was evaporated. The work-up procedure describe above CH3); 13C-NMR (CDCl3) d/ppm:171.7, 169.1, 155.9,
yielded a Z-NCan-Tyr-OMe (0.11 g, 51 %) and Z-NCav- 155.7, 136.1, 130.2, 129.2, 128.9, 127.6, 127.1, 115.8,
Tyr-OMe (0.14 g, 55 %) as an oils, respectively. 23: MS- 79.5, 75.5, 66.4, 63.3, 58.8, 37.3, 28.4; IR (KBr) cm-1
:
ES, m/z: 432 [M?] (431.44); Anal. Calcd. for C21H25N3O7: 3300–2920, 1698, 1535, 1451, 1410, 1332, 1259, 1134,
C, 58.46; H, 5.84; N, 9.74; O, 25.96. Found: C, 58.33; H, 1090, 975, 910, 883; 26: MS-ES, m/z: 516 [M?] (515.56);
1
5.87; N, 9.79; O, 26.01. H-NMR (CDCl3) d/ppm: 8.03 (s, Anal. Calcd. for C25H33N5O7: C, 58.24; H, 6.45; N, 13.58;
2H, NH), 7.47 (m, 2H, Ar), 7.38 (m, 3H, Ar), 7.12 (m, 2H, O, 21.72. Found: C, 58.29; H, 6.40; N, 13.61; O, 21.70.1H-
Ar), 6.7 (d, 2H, Ar), 5.35 (s, 1H, Ph-OH), 5.09 (s, 2H, NMR (CDCl3) d/ppm: 8.58 (s, 3H, Gu), 8.03 (s, 2H, NH),
PhCH2), 4.62 (m, 1H, CH), 4.55 (m, 1H, CH), 4.16 (d, 2H, 7.47 (m, 2H, Ar), 7.38 (m, 3H, Ar), 7.12 (m, 2H, Ar), 6.7
CH2), 3.68 (s, 3H, CH3), 3.23 (d, 2H, CH2), 2.0 (s, 2H, (d, 2H, Ar), 5.35 (s, 1H, Ph-OH), 5.34 (s, 2H, PhCH2), 4.92
NH2); 13C-NMR (CDCl3) d/ppm: 171.5, 170.4, 155.9, (m, 1H, CH), 4.26 (d, 2H, CH2), 3.98 (m, 1H, CH), 3.44 (d,
155.7, 136.1, 130.2, 129.2, 128.9, 127.6, 127.1, 115.8, 76, 2H, CH2), 2.0 (s, 2H, NH), 1.38 (s, 9H, CH3); 13C-NMR
66.8, 56.7, 55.9, 51.9, 36.8; IR (KBr) cm-1: 3371, 3062, (CDCl3) d/ppm:171.7, 169.1, 158.5, 155.9, 155.7, 136.1,
2950, 2424, 1750, 1582, 1454, 1480, 1368, 1255, 1122, 130.2, 129.2, 128.9, 127.6, 127.1, 115.8, 79.5, 73.1, 66.4,
933, 762; 24: MS-ES, m/z: 474 [M?] (473.48); Anal. 63.3, 58.8, 37.3, 28.4; IR (KBr) cm-1: 3350–2928, 1640,
Calcd. for C22H27N5O7: C, 55.81; H, 5.75; N, 14.79; O, 1592, 1404, 1253, 1155, 1062, 974, 879, 803.
23.65. Found: C, 55.72; H, 5.7; N, 14.72; O, 23.86.1H-
NMR (CDCl3) d/ppm: 8.58 (s, 3H, Gu), 8.03 (s, 2H, NH), Synthesis of Z-NCan-Tyr-OH (2-(3-(aminooxy)-2-
7.47 (m, 2H, Ar), 7.38 (m, 3H, Ar), 7.12 (m, 2H, Ar), 6.7 (((benzyloxy)carbonyl)amino)propanamido)-3-(4-
(d, 2H, Ar), 5.35 (s, 1H, Ph-OH), 5.09 (s, 2H, PhCH2), 4.62 hydroxyphenyl)propanoic acid) (27), Z-NCav-Tyr-OH
(m, 1H, CH), 4.55 (m, 1H, CH), 4.16 (d, 2H, CH2), 3.68 (s, (2-(2-(((benzyloxy)carbonyl)amino)-3-
3H, CH3), 3.23 (d, 2H, CH2), 2.0 (s, 2H, NH); 13C-NMR (guanidinooxy)propanamido)-3-(4-hydroxyphenyl)-
(CDCl3) d/ppm:171.5, 170.4, 158.5, 155.9, 155.7, 136.1, propanoic acid) (28), Boc-Tyr-NCan-OH (3-(aminooxy)-2-
130.2, 129.2, 128.9, 127.6, 127.1, 115.8, 73.6, 66.8, 56.7, (2-((tert-butoxycarbonyl)amino)-3-(4-hydroxyphenyl)-
55.9, 51.9, 36.8; IR (KBr) cm-1: 3000–2356, 1692, 1535, propanamido)propanoic acid) (29), and Boc-Tyr-NCav-
1457, 1333, 1255, 1160, 1073, 983, 899, 704.
OH (2-(2-((tert-butoxycarbonyl)amino)-3-(4-
hydroxyphenyl)propanamido)-3-
Synthesis of Boc-Tyr-NCan-OBzl (benzyl 3-(aminooxy)-2-
(2-((tert-butoxycarbonyl)amino)-3-(4-hydroxyphenyl)
propanamido)propanoate) (25) and Boc-Tyr-NCav-OBzl
(benzyl 2-(2-((tert-butoxycarbonyl)amino)-3-(4-
hydroxyphenyl)propanamido)-3-
(guanidinooxy)propanoic acid) (30)
The peptide 23 (24, 25, 26) (0.2 mM) was dissolved in a
mixture of dioxane: water (1: 1, 10 ml), and drop of thy-
molphthalein in MeOH was added. Then a solution of 1 N
NaOH was added dropwise until complete saponification
(blue color of the solution was constant). The dioxane was
(guanidinooxy)propanoate) (26)
A
solution of 2HBr.NCan-OBzl (2HBr.NCav-OBzl) evaporated, and the residue was extracted with EtOAc
(1.1 mM) and DIPEA (0.4 ml, 2.2 mM) in 5 ml DMF was (3 9 10 ml), acidified to pH 3 with dry NaHSO4 and
added to a solution of Boc-Tyr-OSu (0.42 g, 1.1 mM) in extracted again with EtOAc (3 9 10 ml). Combined
5 ml DMF. Reaction was carried out at room temperature organic layers were washed with water, dried over anhy-
for 24 h. After complete the reaction 10 ml of water was drous Na2SO4, filtered and the EtOAc was evaporated. The
added followed by extraction with CHCl3 (3 9 10 ml). work-up procedure describe above yielded a Z-NCan-Tyr-
Combined organic layers were washed consequently with OH (0.059 g, 72 %), Z-NCav-Tyr-OH (0.087 g, 68 %),
5 % NaHCO3 (3 9 10 ml), 5 % NaHSO4 (3 9 10 ml), and Boc-Tyr-NCan-OH (0.02 g, 23 %), and Boc-Tyr-NCav-
brine, dried over anhydrous Na2SO4 and CHCl3 was OH (0.04 g, 44 %), respectively. 27: MS-ES, m/z: 418
evaporated. The work-up procedure describe above yielded [M?] (417.41); Anal. Calcd. for C20H23N3O7: C, 57.55; H,
a 0.35 g (67 %) of Boc-Tyr-NCan-OBzl and 0.37 g (65 %) 5.55; N, 10.07; O, 26.83. Found: C, 57.50; H, 5.57; N,
of Boc-Tyr-NCav-OBzl, respectively. 25: MS-ES, m/z: 474 10.11; O, 26.82.1H-NMR (CDCl3) d/ppm: 11.0 (s, 1H,
[M?] (473.52); Anal. Calcd. for C24H31N3O7: C, 60.88; H, OH), 8.03 (s, 2H, NH), 7.47 (m, 2H, Ar), 7.38 (m, 3H, Ar),
6.60; N, 8.87; O, 23.65. Found: C, 60.82; H, 6.67; N, 8.85; 7.12 (m, 2H, Ar), 6.7 (d, 2H, Ar), 5.35 (s, 1H, Ph-OH), 5.09
O, 23.66.1H-NMR (CDCl3) d/ppm: 8.03 (s, 2H, NH), 7.47 (s, 2H, PhCH2), 4.72 (m, 1H, CH), 4.62 (m, 1H, CH), 4.16
(m, 2H, Ar), 7.38 (m, 3H, Ar), 7.12 (m, 2H, Ar), 6.7 (d, 2H, (d, 2H, CH2), 3.12 (d, 2H, CH2), 2.0 (s, 2H, NH2); 13C-
Ar), 5.35 (s, 1H, Ph-OH), 5.34 (s, 2H, PhCH2), 4.92 (m, NMR (CDCl3) d/ppm: 174.7, 170.4, 155.9, 155.7, 136.1,
1H, CH), 3.44 (s, 2H, PhCH2), 4.26 (d, 2H, CH2), 3.98 (m, 130.2, 129.2, 128.9, 127.6, 121.1, 115.8, 76.0, 66.8, 59.2,
123

Med Chem Res
55.9, 36.5; IR (KBr) cm-1: 3380–2880, 1746, 1528, 1403,
1253, 1176, 1068, 934, 715; 28: MS-ES, m/z: 460 [M?]
(459.45); Anal. Calcd. for C21H25N5O7: C, 54.90; H, 5.48;
N, 15.24; O, 24.38. Found: C, 54.93; H, 5.42; N, 15.28; O,
24.37.1H-NMR (CDCl3) d/ppm: 11.0 (s, 1H, OH), 8.58 (s,
3H, Gu), 8.03 (s, 2H, NH), 7.47 (m, 2H, Ar), 7.38 (m, 3H,
Ar), 7.12 (m, 2H, Ar), 6.7 (d, 2H, Ar), 5.35 (s, 1H, Ph-OH),
5.09 (s, 2H, PhCH2), 4.72 (m, 1H, CH), 4.62 (m, 1H, CH),
4.16 (d, 2H, CH2), 3.12 (d, 2H, CH2), 2.0 (s, 2H, NH); 13C-
NMR (CDCl3) d/ppm: 174.7, 170.4, 158.5, 155.9, 155.7,
136.1, 130.2, 129.2, 128.9, 127.6, 121.1, 115.8, 73.6, 69.2,
66.8, 55.9, 36.5; IR (KBr) cm-1: 3667–3010, 2930, 1708,
1405, 1257, 1133, 1073, 900, 805; 29: MS-ES, m/z: 384
[M?] (383.40); Anal. Calcd. for C17H25N3O7: C, 53.26; H,
6.57; N, 10.96; O, 29.21. Found: C, 53.29; H, 6.52; N,
10.94; O, 29.25. 1H-NMR (CDCl3) d/ppm: 11.0 (s,
1H,OH), 8.03 (s, 2H, NH), 7.12 (m, 2H, Ar), 6.7 (d, 2H,
Ar), 5.35 (s, 1H, Ph-OH), 4.92 (m, 1H, CH), 4.14 (d, 2H,
CH2), 4.02 (m, 1H, CH), 3.44 (s, 2 h, PhCH2), 2.0 (s, 2H,
NH2), 1.38 (s, 9H, CH3); 13C-NMR (CDCl3) d/ppm: 171.7,
171.0, 155.9, 155.7, 130.2, 129.2, 115.8, 79.5, 75.2, 58.8,
55.5, 37.3, 28.4; IR (KBr) cm-1: 3545–2876, 1644, 1593,
1416, 1179, 1135, 1068, 720; 30: MS-ES, m/z: 426 [M?]
(425.44); Anal. Calcd. for C18H27N5O7: C, 50.82; H, 6.40;
N, 16.46; O, 26.32. Found: C, 50.87; H, 6.43; N, 16.41; O,
14.83; O, 28.24. Found: C, 50.90; H, 6.01; N, 14.87; O,
1
28.22. H-NMR (CDCl3) d/ppm: 11.0 (s, 1H, OH), 8.03
(s, 1H, NH), 7.12 (m, 2H, Ar), 6.7 (d, 2H, Ar), 5.35 (s,
1H, Ph-OH), 5.11 (d, 2H, NH2), 4.72 (m, 1H, CH), 4.16
(d, 2H, CH2), 3.65 (m, 1H, CH), 3.12 (d, 2H, CH2), 2.0 (s,
2H, NH2); 13C-NMR (CDCl3) d/ppm: 174.7, 171.7, 155.7,
130.2, 129.2, 115.8, 78.8, 59.2, 52.0, 36.5; IR (KBr)
cm-1: 3550–2960, 1752, 1638, 1403, 1222, 1160, 1079,
898, 738; 11: MS-ES, m/z: 338 [M?] (337.33); Anal.
Calcd. for C14H19N5O5: C, 48.00; H, 5.89; N, 21.53; O,
1
24.59. Found: C, 47.95; H, 5.92; N, 21.55; O, 24.58. H-
NMR (CDCl3) d/ppm: 11.0 (s, 1H, OH), 8.56 (s, 3H, Gu),
8.03 (s, 1H, NH), 7.12 (m, 2H, Ar), 6.7 (d, 2H, Ar), 5.35
(s, 1H, Ph-OH), 5.11 (d, 2H, NH2), 4.72 (m, 1H, CH),
4.16 (d, 2H, CH2), 3.65 (m, 1H, CH), 3.12 (d, 2H, CH2),
2.0 (s, 2H, NH); 13C-NMR (CDCl3) d/ppm: 174.7, 171.7,
158.5, 155.7, 130.2, 129.2, 115.8, 76.4, 59.2, 52.0, 36.5;
IR (KBr) cm-1: 3381–2949, 1740, 1528, 1410, 1230,
1165, 1054, 903, 706.
Synthesis of HCl.Tyr-NCan (2-(2-amino-3-(4-
hydroxyphenyl)propanamido)-3-(aminooxy)propanoic acid)
(18) and HCl.Tyr-NCav (2-(2-amino-3-(4-hydroxyphenyl)-
propanamido)-3-(guanidinooxy)propanoic acid) (19)
1
26.29. H-NMR (CDCl3) d/ppm: 11.0 (s,1H, OH), 8.58 (s,
The peptide 29 (30) (0.4 mM) was dissolved in 2 ml 3 M
HCl/EtOAc. During the reaction crystals of the new pro-
ducts were formed. After 1 h reaction was completed and
obtained crystal product was filtered and washed with
diethyl ether. Final peptides were obtained after column
purification (Silicagel 60, CH3CN: H2O, 4:1). The work-up
procedure describe above yielded a 0.09 g (83 %) of
HCl.Tyr-NCan and 0.07 g (81 %) of HCl.Tyr-NCav, res-
pecively. 18: MS-ES, m/z: 284 [M?] (283.28); Anal. Calcd.
for C12H17N3O5: C, 50.88; H, 6.05; N, 14.83; O, 28.24.
3H, Gu), 8.03 (s, 2H, NH), 7.12 (m, 2H, Ar), 6.7 (d, 2H,
Ar), 5.35 (s, 1H, Ph-OH), 4.92 (m, 1H, CH), 4.14 (d, 2H,
CH2), 4.02 (m, 1H, CH), 3.44 (d, 2H, CH2), 2.0 (s, 2H,
NH), 1.38 (s, 9H, CH3); 13C-NMR (CDCl3) d/ppm: 171.7,
171.0, 158.5, 155.9, 155.2, 130.1, 129.2, 115.8, 79.5, 72.8,
58.8, 55.5, 37.3, 28.4; IR (KBr) cm-1: 3320–2876, 1706,
1654, 1430, 1260, 1142, 1115, 1036, 900, 715.
Synthesis of HCl.NCan-Tyr (2-(2-amino-3-
(aminooxy)propanamido)-3-(4-hydroxyphenyl)propanoic
acid) (10) and HCl.NCav-Tyr (2-(2-amino-3-
(guanidinooxy)propanamido)-3-(4-
1
Found: C, 50.93; H, 6.00; N, 14.85; O, 28.22. H-NMR
(CDCl3) d/ppm: 11.0 (s, 1H,OH), 8.03 (s, 1H, NH), 7.12
(m, 2H, Ar), 6.7 (d, 2H, Ar), 5.35 (s, 1H, Ph-OH), 5.11 (d,
2H, NH2), 4.14 (d, 2H, CH2), 4.02 (m, 1H, CH), 3.95 (m,
1H, CH), 3.44 (s, 2 h, PhCH2), 2.0 (s, 2H, NH2); 13C-NMR
(CDCl3) d/ppm: 171.7, 171.0, 155.7, 130.2, 129.2, 115.8,
75.2, 56.0, 55.5, 38.7; IR (KBr) cm-1: 3300–2876, 1679,
1542, 1401, 1266, 1150, 1078, 895, 711; 19: MS-ES, m/z:
338 [M?] (337.33); Anal. Calcd. for C13H19N5O5: C,
48.00; H, 5.89; N, 21.53; O, 24.59. Found: C, 47.98; H,
hydroxyphenyl)propanoic acid) (11)
The peptide 25 (26) (0.1 mM) was dissolved in 1 ml
CH3COOH and HBr/CH3COOH (0.5 ml) was added. The
deprotection continued for 1 h at room temperature. The
solvent was evaporated, and the crude product was treated
three times with MeOH (3 9 20 ml), which was also
evaporated. The obtained HBr-salts of the kyotorphin
analogues were transformed to free bases with Et3N in
CH2Cl2. The CH2Cl2 was evaporated and dipeptides were
treated with 3 M HCl/EtOAc to obtain crystal HCl-salts
of NCan-Tyr and NCav-Tyr, respectively. Final peptides
were obtained after column purification (Silicagel 60,
CH3CN: H2O, 4:1). 10: MS-ES, m/z: 284 [M?] (283.28);
Anal. Calcd. for C12H17N3O5: C, 50.88; H, 6.05; N,
1
5.85; N, 21.57; O, 24.60. H-NMR (CDCl3) d/ppm: 11.0
(s,1H, OH), 8.56 (s, 3H, Gu), 8.03 (s, 1H, NH), 7.12 (m,
2H, Ar), 6.7 (d, 2H, Ar), 5.35 (s, 1H, Ph-OH), 5.11 (d, 2H,
NH2), 4.14 (d, 2H, CH2), 4.02 (m, 1H, CH), 3.95 (m, 1H,
CH), 3.44 (d, 2H, CH2), 2.0 (s, 2H, NH); 13C-NMR
(CDCl3) d/ppm: 171.7, 171.0, 158.5, 155.7, 130.2, 129.2,
72.8, 56.0, 55.5, 38.7; IR (KBr) cm-1: 3300–2887, 1647,
1404, 1158, 1075, 884, 714.
123

Med Chem Res
Activity assays
activity, proteolytic stability, and bioavailability. Our pre-
vious efforts were focused on the preparation and the
characterization of unnatural amino acids, particularly
those containing a basic functionality (amino, oxyamino,
sulfoamino, oxyguanidinium and sulfoguanidinium) in the
side chain. We synthesized numerous unnatural amino
acids structural analogues of arginine such as: sulfoargi-
nines (sArg, NsArg); canavanines (Cav, NCav), and of
lysine (sLys, HsLys), which demonstrated certain noci-
ceptive effects (Videnov et al., 1993; Pajpanova et al.,
1997; Dzimbova et al., 2011). For instance, in our previous
studies (Brakadanska et al., 2001) we demonstrated that
L-Cav (at a dose 20 lg/rat, i.c.v) exerted strong naloxone-
reversible antinociceptive effects Fig. 3).
Animals
Male Wistar rats (180–200 g) were used and kept under
normal conditions at ambient room temperature (22 °C).
The experimental procedures were carried out in accor-
dance with the institutional guidance and general recom-
mendations on the use of animal for scientific purposes.
Paw-pressure test (PP)
Changes in the mechanical nociceptive threshold of rats
were measured using an analgesimeter (Ugo Basile)
(Randall and Selitto, 1957). The pressure was applied to
the dorsal surface of rat hind paw and the pressure
(g) required eliciting nociceptive responses such as squeak,
and struggle was taken as mechanical nociceptive thresh-
old. A cut-off value of 500 g was used to prevent paw
damage.
We examined for possible analgesic activity the syn-
thesized sulfoarginine, its derivatives and canavanine in
rats by PP nociceptive test. All amino acids were applied
intraperitoneally (i.p.) at a dose of 1 mg kg-1. The inves-
tigations started 15 min. after injection of amino acids, and
all amino acid analogues showed significant analgesic
effects compared to the control (Fig. 3). Tyr-NsArg-NH2,
Tyr-NsArg-OBzl (all at dose 5 mg kg-1) estimated by
paw-pressure test (Bocheva et al., 2006). Data are pre-
sented as mean SEM.
Data analysis
The results were statistically assessed by an analysis of
variance (ANOVA). All results are expressed as
mean SEM. One-way analysis of variance was used to
verify the statistical significance at P \ 0.05 between the
treated and control groups.
To illustrate the impact of this non-proteinogenic amino
acid on bioactivity, we synthesized a number of analogues
modified in both positions (Fig. 4) by introducing Cav
instead Arg into the sequence. We also studied the antin-
ociceptive activity analgesic effects of Tyr-Cav, Tyr(Cl2)-
Cav on the most frequently used models based on tests with
thermal (HP, TF) and mechanical irritation (PP). Changes
in the nociceptive effects of the peptides applied (i.c.v.) at a
dose of 20 lg rat-1 in male Wistar rats were examined by
(TF) and (HP) tests. The obtained results showed that Kyo,
Tyr-Cav and Tyr(Cl2)-Cav significantly increased the
latency in both used nociceptive tests. Tyr-Cav exerted a
strong analgesic action by tail-flick test, and the effect is a
naloxone-reversible. The effects of Tyr-Cav were more
pronounced compared to Kyo (Bocheva et al., 1996).
Further we investigate the possible analgesic activity of
synthesized Kyo analogues containing NsArg and deriva-
tives in rats (Bocheva et al., 2006). The antinociceptive
effects of all these dipeptides (NsArg-Tyr, Tyr-NsArg-NH2
and Tyr-NsArg-Bzl) were evaluated after i.p. administra-
tion in rats. All peptides at a dose of 5 mg kg-1 estimated
by PP test showed naloxone-reversible analgesic activity
versus control and Kyo. The most active compound Tyr-
NsArg-Bzl was found to increase significantly the pain
threshold versus native peptide Kyo on 15th minute. On
30th minute only Tyr-NsArg has analgesic effect compared
to the control and Kyo. In time the analgesic effects of
newly synthesized analogues quickly decreased and on the
45th minute they completely vanished.
Computational tools
In order to perform computational studies the different
software was used in the present work: —crystal structure
of the l-opioid receptor was obtained from RCSB (PDB id:
4dkl, http://www.rcsb.org/pdb/home/home.do); —ligand
preparation was done with Avogadro (an open-source
molecular builder and visualization tool —Version 1.0.3,
http://avogadro.openmolecules.net/); —docking studies
were performed by using GOLD 5.1 (Genetic Optimization
for Ligand Docking, Jones et al., 1997), run on Scientific
LINUX 5.5 operating system; —for generation figures
Molegro Molecular Viewer (http://molegro.com/index.
php) was used.
Results and discussion
We were interested in studying and comparing the effects
of the replacement of Arg with various unnatural amino
acids on the structural properties and on the biological
activity of kyotorphin.
Non-proteinogenic, unnatural a-amino acids have been
widely used as modifiers of peptides to enhance biological
123

Med Chem Res
Fig. 3 Effects of kyotorphin
analogues estimated by paw-
pressure test. (L-Arg, L-Orn,
L-Cit, L-Can and L-Cav—i.c.v
administration, all at dose
20 lg/kg, Brakadanska et al.,
2001; Cys(O2NH2), NsArg,
NsArg-NH2, NsArg-OBzl,
L-Cav and L-Can—i.p.
administration, all at dose 1 mg/
kg, and NsArg, NsArg-Tyr, Tyr-
NsArg, Tyr-NsArg-NH2, Tyr-
NsArg-OBzl—i.p.
administration, all at dose 5 mg/
kg, Bocheva et al., 2006). Data
are presented as mean SEM
Fig. 4 Design of Kyo
analogues
It is well documented that the mammalian brain contains
all the urea cycle intermediates, whereas enzymes partici-
pating in the conversion of L-ornithine (L-Orn) to L-cit-
rulline (L-Cit) are absent, resulting in an incomplete urea
cycle (Garthwaite, 1991). On the other hand, in our pre-
vious in vivo experiments we demonstrated that L-Cav,
L-Cit and L-Orn exerted antinociceptive effects (Brak-
adanska et al., 2001; Bocheva et al., 1996).
structure, four analogues with the following structural
formulas were obtained: Tyr-NCav; Tyr-NCan; NCav-Tyr
and NCan-Tyr.
The peptides were synthesized by a standard solution
peptide synthesis. For the synthesis of reverse-kyotorphin
analogues NCan-Tyr and NCav-Tyr condensation of
Z-NCan, respectively Z-NCav and Tyr-OMe was applied,
using method of mixed anhydrides (Fig. 5a).
All this and the fact that L-norcanaline (NCan) and
L-norcanavanine (NCav) are structural analogues of orni-
thine and canavanine, respectively, stimulated us to syn-
thesize new analogues of kyotorphin which were expected
to possess analgesic activity. Namely, NCan and NCav
were introduced into the peptide chain of kyotorphin. Due
to direct modification in both positions of the parent
The hydrolysis of methyl ester was done by 1 N NaOH
in water: dioxane mixture (1:1), followed by acidolysis of
Z-group with HBr/CH3COOH. Final peptides were
obtained in good yields after column purification (Silicagel
60, CH3CN: H2O, 4:1).
Modified in position 2 kyotorphins were synthesized
using method of activated esters (Fig. 5b). The Z-Tyr-OSu
123

Med Chem Res
B
AA
A
Tyr
Z
Z
OH
AA
Tyr
Boc
Boc
Boc
OH
OBzl
OBzl
OBzl
OMe
OMe
Z
Z
OH
HCl.H
OSu
2HBr.H
i
i
25, 26
23,
ii
ii
29, 30
18, 19
Z
OH
OH
Boc
H
OH
OH
27,
10,
iii
iii
H
AA = NCan, NCav
AA = NCan, NCav,
i) DIPEA, DMF, room temperature;
ii) 1N NaOH, dioxane:H2O (1:1);
i) Piv-Cl, NMM, Et3N, DMF, -10oC;
ii) 1N NaOH, dioxane:H2O (1:1);
iii) HBr/AcOH, AcOH
iii) 3 M HCI/EtOAc
Fig. 5 Synthesis of NCan-Tyr and NCav-Tyr (a) and Tyr-NCan and Tyr-NCav (b)
reacted with benzyl esters of NCan or NCav prepared in
advance. Reaction time was about 24 h fully protected
peptides were obtained in 63 and 54 % yields respectively.
Final peptides were obtained in good yields after column
purification (Silica gel 60, CH3CN: H2O, 4:1).
paw-pressure tests for all compounds described above.
Docking was carried out with GOLD 5.1 software. It uses
genetic algorithm and considers full ligand conformational
flexibility and partial protein flexibility. For docking
studies the crystal structure of l-opioid receptor, published
in RCSB was used. It was published (Befort et al., 1996)
that the binding site for opioid receptors was defined as
Each peptide was analyzed for purity on high-perfor-
mance liquid chromatography (HPLC) and characterized
1
by H- and 13C-NMR, IR-spectrometry, and high-resolu-
residues within 10 A radius of aspartic acid of third trans
˚
tion mass spectra with a Fissons-Triple Quadrupol-ES mass
spectrometer.
membrane domain, which is involved in the most crucial
interaction. In the case of l-opioid receptor this is Asp147.
GoldScore algorithm was used and Fitness scoring function
was calculated for each ligand. All Fitness function’s val-
ues are listed in the Table 1.
Conventional IR spectroscopy is not very informative
for peptides but NMR spectra provide the most complete
information on the structure of the obtained compounds.
NCan is a structural analogue of the amino acid ornithine
and NCav—of arginine, respectively. A comparison with
published data in Biological Magnetic Resonance Data
Bank (icnm.cerm.inifi.it) for natural amino acids was
made. The shift of the protons of the d-amino group of
ornithine in 1H-NMR is at 1.8 ppm. The effect of the
bound oxygen atom is low and the shift of the bands of the
analogues containing NCan in lower field is negligible. The
chemical shift is at about 2 ppm.
There is a good correlation between number of interactions
between l-opioid receptor and ligand, and its biological
activity. The most potent compounds according to data in the
Table 1, are Tyr-NsArgOBzl, Tyr-Cav, L-NAME, Tyr(Cl2)-
Cav. They interact with the receptor sterically, electrostati-
cally, and by forming hydrogen bonds. In those cases number
of interactions was more than 12. The ligands with the lowest
potency [L-Orn, L-Cit, Cys(SO2NH2)] interacted weaker with
receptor with not more than 6 interacting sites. Examples of
interactions were shown on Fig. 6.
However, the influence of the oxygen atom in the ana-
logues containing NCav in their molecules is significant. In
the spectrum of arginine the protons of the guanidinium
group are shifted at 1.67–1.7 ppm, while the protons of the
oxyguanidinium group are shifted to the lower field at
about 8 ppm under the influence of the oxygen atom.
The chemical shift of the C-atoms of the guanidinium
group of arginine is at 36.2 ppm in 13C-NMR, while under
the influence of oxygen atom this shift for the C-atom of
the oxyguanidinium group is in lower filed at about
158 ppm.
Exploring docking results, almost all studied ligands
interacted with residue Asp147 from the l-opioid receptor
sequence. This residue was involved in forming hydrogen
bonds with appropriate functional group of the ligand. The
neighbor residue Tyr148 played important role in ligand-
receptor interaction also, either by forming hydrogen bond
with its OH group, or by p-p interactions with ligand.
Thekyotorphinfor exampleformed6 hydrogenbonds with
residues in the receptor pocket: its carboxylic group interacted
with three residues Gln124, Asp147, and Tyr326; the free
amino group interacted by hydrogen bonding with hydroxyl
group of Tyr148; and the guanidinium group formed two
hydrogen bonds with Gln124 (Fig. 7a). One of the newly
synthesizedkyotorphinanalogues(NCav-Tyr)interactedwith
Further, we tried to find relationship between structure
and activity, and to predict the biological effect of the
newly synthesized kyotorhin analogues 18 and 19, using a
docking sofware. In this regard, we collected data from
123

Med Chem Res
l-opioid receptor by forming 7 hydrogen bonds. The oxy-
guanidinium group formed two hydrogen bonds with Tyr148,
the free amino group was connected with Asp147, the free
carboxylic group interacted by forming 3 hydrogen bonds
with Asp147, Tyr326, and Gln124, respectively and the
hydroxyl group was hydrogen bonded with Cys217 residue
(Fig. 7b).
Table 1 Values from paw-pressure test from in vivo studies and
fitness function from docking studies for all studied compounds
Number
Compounds
PP, g cm2
Fitness
1
Cys(SO2NH2)
D-Kyo
130
41.37
64.72
69.38
44.32
54.51
45.71
54.00
36.19
40.56
57.92
64.77
49.42
52.02
57.51
61.02
65.23
68.83
55.49
63.21
66.67
65.83
77.15
2
241a
265b
204
3
Kyo
4
L-Can
Significant relationship was found between external
energy of the bonded ligands and their biological effect. In
this context, as less is this energy, as higher is the bio-
logical effect of investigated compound.
5
L-Cav
188
6
L-Cit
151
283b
7
L-NAME
L-NCan
8
274
Correlations of docking data and results from the in vivo
experiments were performed with GraphPad Prism 3.0. The
pain threshold in g cm2 was compared with the results from
docking (Fitness scoring function) in order to find corre-
lation. Good positive Pearson’s correlation was obtained
between Fitness scoring function from GOLD docking
procedure with threshold at 15th minute of paw-pressure
test (Fig. 8). As higher is a value for Fitness scoring
function as higher is biological effect of the investigated
compound. Value for Fitness scoring function could be
easily obtained from docking results for any compound
with l-opioid receptor. Using this model it is simply to
calculate the values of paw-pressure threshold and predict
the biological effect.
9
L-Orn
121
10
11
12
13
14
15
16
17
18
19
20
21
22
NCan-Tyr
NCav-Tyr
NsArg
235
295
222c
NsArgNH2
NsArgOBzl
NsArg-Tyr
Tyr-Cav
208
186
260b
308b
280b
220
Tyr(Cl2)-Cav
Tyr-NCan
Tyr-NCav
Tyr-NsArg
Tyr-NsArgNH2
Tyr-NsArgOBzl
280
252c
267c
340c
As well as docking was performed with the l-opioid
receptor structure, and there is correlation between in vivo
results and Fitness function, assumption made from us
before could be true. In other words, if the kyotorphin
receptor exist its binding pocket, is the similar as in the
l-opioid receptor.
In red are presented predicted values of threshold of the newly syn-
thesized kyotorphin analogues
a
Results of previous research (Dzambazova et al., 2011)
b
Results of previous research (Dzambazova-Maximova et al., 2003)
c
Results of our previous research (Bocheva et al., 2006)
Fig. 6 Interactions in the binding pocket of l-opioid receptor of Tyr-NsArg-OBzl (a) and L-Orn (b). Colors in the pictures red—steric and
electrostatic interactions, blue—hydrogen bonds (Color figure online)
123

Med Chem Res
Conclusion
In summary, four new analogues of kyotorphin containing
NCan and NCav were synthesized using well-known
methods of peptide synthesis in solution. We have dem-
onstrated that arginine mimetics, as well as amino acids
from urea cycle exerted strong naloxone-reversible antin-
ociceptive effects in in vivo test.
The molecular docking procedure was applied to
determine the possible interactions for our ligands and the
receptor. From docking studies we could conclude that
there is evidence to assume that even kyotorphin receptor
exists its binding pocket, is not very different from those of
l-opioid receptor. By means of the docking studies we
could predict biological effects of newly synthesized
compounds, and we could propose structures with higher
biological activity.
Acknowledgments This work has been supported by Grant MY-
FS-13 and DVU 01/197 of the National Science Fund of the Ministry
of Education and Science of Republic of Bulgaria and COST Action
CM0801 Project DO 02-135/31.07.2009.
Conflict of interest The authors declare that they have no conflict
of interest.
Fig. 7 Hydrogen bond formed in binding pocket of l-opioid receptor
with kyotorphin (a) and NCav-Tyr (b)
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