Discovery of kynurenines containing oligopeptides as potent opioid receptor agonists

Article abstract of DOI:10.3390/biom10020284

Kynurenine (kyn) and kynurenic acid (kyna) are well-defined metabolites of tryptophan catabolism collectively known as “kynurenines”, which exert regulatory functions in host-microbiome signaling, immune cell response, and neuronal excitability. Kynurenine containing peptides endowed with opioid receptor activity have been isolated from natural organisms; thus, in this work, novel opioid peptide analogs incorporating L-kynurenine (L-kyn) and kynurenic acid (kyna) in place of native amino acids have been designed and synthesized with the aim to investigate the biological effect of these modifications. The kyna-containing peptide (KA1) binds selectively the μ-opioid receptor with a Ki = 1.08 ± 0.26 (selectivity ratio μ/δ/? = 1:514:10000), while the L-kyn-containing peptide (K6) shows a mixed binding affinity for μ, δ, and ?-opioid receptors, with efficacy and potency (Emax = 209.7 + 3.4%; LogEC50 = ?5.984 + 0.054) higher than those of the reference compound DAMGO. This novel oligopeptide exhibits a strong antinociceptive effect after i.c.v. and s.c. administrations in in vivo tests, according to good stability in human plasma (t1/2 = 47 min).

Full text of DOI:10.3390/biom10020284

biomolecules
Article
Discovery of Kynurenines Containing Oligopeptides
as Potent Opioid Receptor Agonists
Edina Szu˝cs 1,2, Azzurra Stefanucci 3,*, Marilisa Pia Dimmito 3, Ferenc Zádor 1,
Stefano Pieretti 4 , Gokhan Zengin 5 , László Vécsei 6, Sándor Benyhe 1, Marianna Nalli 7 and
Adriano Mollica 3
1
Institute of Biochemistry, Biological Research Center, Hungarian Academy of Sciences, Temesvári krt. 62.,
H-6726 Szeged, Hungary; szucs.edina@brc.hu (E.S.); zador.ferenc@brc.hu (F.Z.); benyhe.sandor@brc.hu (S.B.)
Doctoral School of Theoretical Medicine, Faculty of Medicine, University of Szeged, Dómtér 10,
2
H-6720 Szeged, Hungary
3
Department of Pharmacy, University of Chieti-Pescara “G. d’Annunzio”, Via dei Vestini 31, 66100 Chieti,
Italy; marilisa.dimmito@unich.it (M.P.D.); a.mollica@unich.it (A.M.)
National Center for Drug Research and Evaluation, Istituto Superiore di Sanità, Viale Regina Elena 299,
4
00161 Rome, Italy; stefano.pieretti@iss.it
Department of Biology, Science Faculty, Selcuk University, 42250 Konya, Turkey;
5
gokhanzengin@selcuk.edu.tr
MTA-SZTE Neuroscience Research Group, Department of Neurology, Interdisciplinary Excellence Centre,
6
Faculty of Medicine, University of Szeged, H-6725 Szeged, Hungary; vecsei.laszlo@med.u-szeged.hu
Laboratory affiliated with the Institute Pasteur Italy-Cenci Bolognetti Foundation, Department of Drug
7
Chemistry and Technologies, Sapienza University of Rome, Piazzale Aldo Moro 5, I-00185 Roma, Italy;
marianna.nalli@uniroma1.it
*
Correspondence: a.stefanucci@unich.it
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 27 December 2019; Accepted: 6 February 2020; Published: 12 February 2020
Abstract: Kynurenine (kyn) and kynurenic acid (kyna) are well-defined metabolites of tryptophan
catabolism collectively known as “kynurenines”, which exert regulatory functions in host-microbiome
signaling, immune cell response, and neuronal excitability. Kynurenine containing peptides endowed
with opioid receptor activity have been isolated from natural organisms; thus, in this work, novel
opioid peptide analogs incorporating L-kynurenine (L-kyn) and kynurenic acid (kyna) in place of
native amino acids have been designed and synthesized with the aim to investigate the biological
effect of these modifications. The kyna-containing peptide (KA1) binds selectively the
receptor with a Ki = 1.08 0.26 (selectivity ratio = 1:514:10,000), while the L-kyn-containing
peptide (K6) shows a mixed binding affinity for -opioid receptors, with efficacy and potency
max = 209.7 + 3.4%; LogEC50 5.984 + 0.054) higher than those of the reference compound DAMGO.
µ-opioid
±
µ/δ/κ
µ, δ, and κ
(
E
=
−
This novel oligopeptide exhibits a strong antinociceptive effect after i.c.v. and s.c. administrations in
in vivo tests, according to good stability in human plasma (t1/2 = 47 min).
Keywords: peptides; kynurenines; binding affinity; µ-opioid receptor; pharmacophore; G-protein
activation
1. Introduction
The kynurenine pathway (KP) is an essential part of the tryptophan metabolism in mammalian
tissues, where it is responsible for the formation of two principal metabolites, namely, L-kynurenine
(kyn) and kynurenic acid (kyna). Kyn can arise in peptides and proteins by post/translational
modifications or direct oxidation of tryptophan. It is present in lens crystallins, human Cu2+/Zn2+
dismutase, milk proteins, actin oxidized in vivo, and several bioactive compounds produced by bacteria
Biomolecules 2020, 10, 284; doi:10.3390/biom10020284
www.mdpi.com/journal/biomolecules

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and marine organisms [
1]. Daptomycin is a cyclic kyn-containing lipodepsipeptide approved by the
Food and Drug Administration (FDA), isolated from Streptomyces roseoporus used in the treatment of
Gram-positive pathogen skin infections [ ]. Cyclomontanin B isolated from Annona montana exhibits
promising anti-inflammatory activity [ ]. The kyn-containing peptide FP-Kyn-L-NH2 is the minor
2
3
component of Australian red tree frog skin Litoria rubella collected in central Australia, endowed with
opioid activity at 10−7 M (Figure 1) [4].
Figure 1. Natural bioactive compounds containing kyn residue.
The occurrence of kyn in natural products suggests a possible specificity towards their biological
targets. The enzymes of the human kynurenine pathway are expressed in different tissues and cell types
throughout the body [
1]. In humans, the majority of kyn is excreted by urine; thus, its bioavailability
increases according to the tryptophan flux downstream of the KP [
5
]. Kyn is able to penetrate the
central nervous system (CNS) by transport across the blood–brain barrier (BBB), but it is also produced
locally [6].
Kyna has been originally discovered in canine urine, but a huge amount has been measured in
the gut, bile, human saliva, synovial and amniotic fluid; it has also been detected in food products
such as broccoli, some potatoes, and honeybee products [
on the N-methyl-D-aspartate (NMDA) receptor and other glutamate receptors such as AMPA and
kainate receptors [ ]. Kyna is also found to have an agonistic effect on the G protein coupled receptor
GPR35 [ 10], which can be found in various tissues and organs such as gastrointestinal tract, liver,
1]. Kyna possesses an antagonistic effect
7,8
9,
immune system, central nervous system, and cardiovascular system [11]. NMDA receptors are essential
for the control of the glutamatergic work at the CNS; in contrast to the kainate and AMPA receptors,
the NMDA mediates the influx of Ca2+ ions into neurons, playing an important role in synaptic
plasticity, memory, and learning [
severe cell damage, and apoptosis of neurons, which are strongly related to neurodegenerative and
CNS disorders such as depression, stroke, ischemia, and neuropathic pain [10 12]. Different therapeutic
7,8]. Overactivation of NMDA receptors can lead to excitotoxicity,
–
approaches based on thekynurenine pathway have been postulated to circumvent this problem, such
as the use of kynurenic acid prodrugs or analogs able to penetrate more readily than the parent
compounds or the involvement of ascorbate conjugation to promote the interaction of kyna with
SVCT2 transport protein [13–15]. Intracisternal kyna attenuates formalin-induced nociception in
animals together with antagonist activity at the glycine binding site of NMDA, which is associated with
analgesic properties in rats [16]. At the peripheral sites, kyna decreases the nociceptive behavior in the
tail flick and hot plate tests [16]. Administration of L-kyn and probenecid together with kyna analogs
inhibits NMDA receptors in animal models of trigeminal activation and sensitization [17]. Noteworthy,
kyna and its analogs are able to act on second-order neurons, decreasing mechanical allodynia and pain
sensitivity in different animal pain models [18]. Considering the presence of kyn residue in natural
peptide sequences and the important role exerted by both kynurenines at the CNS [19–21], we plan to
investigate the biological consequences of the insertion of these residues in opioid pharmacophoric
sequences. Kyn could be used in place of phenylalanine, considering its aromatic side chain, whereas

Biomolecules 2020, 10, 284
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kyna could be used as C-terminus to mimic an additional aromatic residue. In this preliminary work,
we performed the synthesis and biological screening of six novel kynurenines containing peptides,
aiming to investigate the modifications imposed by the presence of kyn and kyna on the biological
properties of known endogenous and synthetic opioid peptides in vivo and in vitro. Peptide KA1
retains the DAMGO primary sequence, but the OH terminal group is esterified by kynurenic acid.
Peptides K2 and K3 are EM-2 analogs in which the Phe residues in positions 3 and 4 have been
replaced with kyn and kyn C-terminal amides, respectively. Peptides K4–K6 are enkephalin-like
peptides containing kyn in position 5, bearing as C-terminal the methyl ester, acid, and amide group,
respectively. The novel chemical entities were prepared following solution phase peptide synthesis
and were obtained as TFA salts in good overall yields and excellent purities.
2. Materials and Methods
2.1. Chemistry
All reagents and solvents were acquired from Sigma-Aldrich (Milano, Italy). Solution phase
peptide synthesis was applied to prepare the final products KA1, K2–K6 as TFA salts, following
the procedures reported below. Boc-protected intermediates were purified by silica gel column
chromatography where necessary, or by trituration in Et2O. Final products KA1, K2–K6 were purified
by RP–HPLC on a Waters XBridge BEH130 (C18 5.0 m, 250 10 mm column; flow rate of 7 mL/min;
µ
×
Waters Binary pump 1525; eluent: linear gradient of H2O/ACN 0.1% TFA, ranging from 5% to 95%
ACN in 32 min). The purity of the Nα-Boc-protected products was confirmed by NMR analysis on a
Varian Mercury 300 MHz. The purity of all final compounds was assessed by NMR analysis, ESI–LRMS,
and by analytical RP–HPLC (C18-bonded 4.6
H2O/ACN 0.1% TFA, ranging from 5% to 95% ACN in 26 min, recorded at 254, 275, and 213 nm and
was found to be 95%). The mass spectrometry (MS) equipment was composed as follows: LCQ
×
150 mm; flow rate of 1 mL/min; eluent: gradient of
≥
Thermo Finnigan ion trap mass spectrometer (San Jose, CA, USA) with an electrospray ionization
(ESI) source; capillary temperature: 300 ◦C; spray voltage: 4.00 kV; nitrogen (N2) as the sheath and
auxiliary gas.
2.2. General Procedures
2.2.1. Formation of Ethanolamine-Kynurenic Acid Ester
Kynurenic acid (2 mmol, 1 equiv.) was dissolved in DMF (5 mL) stirring in agitation at 0 ◦C, then
a mixture of Boc-N-aminoethanol (2 mmol, 1 equiv.) and DMAP (0.6 mmol, 0.3 equiv.) in DMF (3 mL)
was transferred in the round bottom flask. After 10 min, EDC.HCl (2.2 mmol, 1.1 equiv.) was added to
the reaction mixture in agitation at 0 ◦C for 10 min, then at r.t. overnight. The solvent was removed in
a rotary evaporator and the oily residue was taken up with EtOAc and washed with 5% citric acid
solution (3 times), NaHCO3 s.s. (3 times), and NaCl s.s. (3 times). Organic phases were collected and
dried on Na2SO4 anhydrous, filtered and dried in rotavapor and high vacuum to give a yellow oily
product. The crude product was triturated with Et2O (2 times), the aqueous layer filtered up, and the
white solid product dried in a rotary evaporator and high vacuum.
2.2.2. Coupling Reaction
Boc-protected compound (1.1 equiv.) was dissolved in DMF (5 mL) in an iced-cooled bottom flask,
then EDC.HCl (1.1 equiv.) and HOBt hydrate (1.1 equiv.) were added stirring for 10 min. A solution of
N-terminal free intermediate (1 equiv.) and DIPEA (3.3 equiv.) in DMF (5 mL) was transferred in the
ice-cooled bottom flask at 0 ◦C and allowed to react at r.t. overnight. The solvent was removed in a
rotary evaporator; the oily residue was taken up with EtOAc and washed with 5% citric acid solution
(3 times), NaHCO3 s.s. (3 times), and NaCl s.s. (3 times). Organic phases were collected and dried on
Na2SO4 anhydrous, filtered and dried in a rotavapor and high vacuum to give a yellow oily product.

Biomolecules 2020, 10, 284
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The crude product was triturated with Et2O (2 times), the aqueous layer filtered up and the white solid
product dried in a rotary evaporator and high vacuum.
2.2.3. Amidation
The Boc-protected or free N-terminal compound (1 equiv.) was dissolved in THF (7 mL) stirring at
−
15 ◦C, then NMM (2.5 equiv.) and iBCF (2.1 equiv.) were added to the solution allowing to react for
30 min. Then NH4OH aq. solution (0.21 mL) was added to the reaction mixture at
15 ◦C for 30 min.
−
The reaction mixture was allowed to react at r.t. for 2 h. The solvent was removed in rotavapor and
the solid residue was dissolved in EtOAc washing with 5% citric acid solution (3 times), NaHCO3 s.s.
(3 times), and NaCl s.s. (3 times). Organic phases were collected and dried on Na2SO4 anhydrous,
filtered and dried in a rotavapor and high vacuum to give a yellow oily product. The crude product
was triturated with Et2O (2 times), the aqueous layer filtered up, and the product dried in a rotary
evaporator and high vacuum to give a white solid product.
2.2.4. Saponification
Boc-protected compound (0.1 mmol, 1 equiv.) was dissolved in THF (5 mL) stirring at r.t. then
NaOH 1M (4 equiv.) was added dropwise, allowing it to react for 3 h. The solvent was removed in a
rotavapor; the oily residue was taken up with water and washed with Et2O (2 times). The aqueous
solution was acidified with HCl 1 M until complete precipitation of the solid residue, which was
extracted with EtOAc 3 times. The organic layers were collected, dried on Na2SO4 anhydrous, filtered
and dried in a rotavapor and high vacuum to give a white solid product.
2.3. Synthesis and Characterization
Description of the reaction procedures [22
detail in the Supplementary Materials.
–24] and compounds characterization are reported in
2.4. In Vitro Biological Assays
2.4.1. Chemicals
Tris-HCl, EGTA, NaCl, MgCl2 6H2O, GDP, the GTP analog GTPγS, and the
·
L-tryptophan metabolite kynurenic acid were from Sigma-Aldrich (Budapest, Hungary);
Tyr-D-Ala-Gly-(NMe)Phe-Gly-ol (DAMGO) was purchased from Bachem Holding AG (Bubendorf,
Switzerland); endomorphin-2 (EM-2) was kindly provided by MTA-ELTE Research Group of Peptide
Chemistry (Budapest, Hungary); Ile5,6-deltorphine II (Ile5,6Delt II) was purchased from Isotope
Laboratory of BRC (Szeged, Hungary); and the highly selective KOR agonist diphenethylamine
derivative, HS665 [25], was offered by Dr. Helmut Schmidhammer (University of Innsbruck,
Austria). Naloxone was provided by Endo Laboratories DuPont de Nemours (Wilmington, DE,
USA). The non-competitive NMDA antagonist, (+)-MK 801 maleate (MK-801) and L-kynurenine
(L-kyn) were obtained from Tocris Bioscience (Bristol, UK). A solution of each ligand in water was
stored in 1 mM stock solution at −20 ◦C. The radiolabelled GTP analog, [35S]GTPγS (specific activity:
1000 Ci/mmol), was acquired from Hartmann Analytic (Braunschweig, Germany). [3H]DAMGO [26
(specific activity: 38.8 Ci/mmol), [3H]Ile5,6Delt II (specific activity: 19.6 Ci/mmol), and [3H]HS665 [27
]
]
(specific activity: 13.1 Ci/mmol) were radiolabelled in the Isotope Laboratory of BRC (Szeged, Hungary).
[3H]MK-801 [28] (specific activity: 30 Ci/mmol) was purchased from PerkinElmer (Boston, MA, USA)
and the UltimaGoldTM MV aqueous scintillation cocktail was from PerkinElmer (Boston, MA, USA).
2.4.2. Animal
Male and female Wistar rats and guinea pigs were used for membrane preparations. The animals
were guarded in a temperature-controlled room, ranging from 21 to 24 ◦C, under a 12:12 light and dark
cycle with water and food ad libitum. All housing and experiments were conducted in accordance with

Biomolecules 2020, 10, 284
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the European Communities Council Directives (86/606/ECC) and the Hungarian Act for the Protection
of Animals in Research (XXVIII.tv. 32.§). The total number of animals, as well as their suffering,
was minimized whenever possible.
2.4.3. Preparation of Brain Samples for Binding Assays
Rats and guinea pigs were decapitated, and their brains were quickly removed. The brains were
used for membrane preparation following the procedure reported by Benyhe [29] for binding and
[35S]GTPγS binding experiments, in agreement with the protocol of Zádor et al. [30].
Homogenization of brains was performed in 30 volumes (v/w) of ice-cold 50 mM Tris-HCl pH 7.4
buffer with a Teflon-glass Braun homogenizer at 1500 rpm. The centrifuge was settled at 18,000 rpm for
20 min at 4 ◦C, the resulting supernatant discarded, and the pellet taken up in the original volume of
Tris-HCl buffer. Incubation of homogenate at 37 ◦C for 30 min was performed in a shaking water-bath.
Five volumes of 50 mM Tris-HCl pH 7.4 buffer were used to suspend the final pellet at
[35S]GTP
S binding experiments, the brains were homogenized with a Dounce in 5 volumes (v/w) of
ice-cold TEM (Tris-HCl, EGTA, MgCl2) at
80 ◦C. The protein content of the membrane preparation
−
80 ◦C. For the
γ
−
was determined by the method of Bradford, BSA being used as a standard [31].
2.4.4. Receptor Binding Assays
Functional [35S]GTPγS Binding Experiments
The functional [35S]GTP
γS binding experiments were performed as previously described [32,33].
Briefly the membrane homogenates were incubated at 30 ◦C for 60 min in Tris-EGTA buffer (pH 7.4)
composed of 50 mM Tris-HCl, 1 mM EGTA, 3 mM MgCl2, 100 mM NaCl, containing 20 MBq/0.05 cm3
γ
[35S]GTP S (0.05 nM) and increasing concentrations (10−10 to 10−5 M) of ligands. The experiments
were performed in the presence of excess GDP (30
µM) in a final volume of 1 mL. Total binding was
measured in the absence of test compounds, non-specific binding was determined in the presence of
10 µM unlabeled GTPγS. The reaction was terminated by rapid filtration under vacuum and washed
three times with 5 mL ice-cold 50 mM Tris-HCl (pH 7.4) buffer through Whatman GF/B glass fibers.
The radioactivity of the filters was detected in an UltimaGoldTM MV aqueous scintillation cocktail
with a Packard Tricarb 2300TR liquid scintillation counter.
Binding Experiments
In MOR, DOR, and KOR displacement, aliquots of frozen rat and guinea pig brain membrane
homogenates were thawed and suspended in 50 mM Tris-HCl buffer (pH 7.4); in NMDA displacement,
the Tris-HCl buffer (pH 7.4) contained 100 µM glycine and 100 µM L-glutamic acid. Samples were
incubated in the presence of the unlabeled ligands in increasing concentrations (10−10 to 10−5 M) for at
35 ◦C for 45 min with [3H]DAMGO, for at 35 ◦C for 45 min with [3H]Ile5,6Delt II, at 25 ◦C for 30 min
with [3H]HS665, and at 25 ◦C for 120 min with [3H]MK-801. The non-specific and total binding was
determined in the presence and absence of 10 µM unlabelled naloxone (MOR and DOR), HS665 (KOR),
and MK-801 (NMDA). Radioactivity of the filter disks was measured, as written above.
Data Analysis
Experimental data are presented as means
±
S.E.M. Sigmoid dose-response curves were fitted
with GraphPad Prism 5.0 (GraphPad Prism Software Inc., San Diego, CA, USA). An unpaired t-test
with two-tailed p value was performed to determine the significance level. In the competition binding
assays, the ‘One site competition’ fitting was used to establish the equilibrium binding affinity (Ki
value).

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2.5. In Vivo Tests
2.5.1. Animals
In our experiments, we used CD-1 male mice (Harlan, Italy, 25–30 g) maintained in colony, housed
in cages (7 mice per cage) under standard light/dark cycle (from 7:00 a.m. to 7:00 p.m.), temperature
(21
±
1 ◦C) and relative humidity (60%
±
10%) for at least 1 week. Food and water were available ad
and the
libitum. The Service for Biotechnology and Animal Welfare of the Istituto Superiore di Sanit
à
Italian Ministry of Health authorized the experimental protocol according to Legislative Decree 26/14.
2.5.2. Treatment Procedure
DMSO was purchased from Merck (Rome, Italy). Peptides solutions were freshly prepared using
saline containing 0.9% NaCl and DMSO in the ratio DMSO/saline 1:5 v/v every experimental day. These
solutions were injected at a volume of 10 µL/mouse for intracerebroventricular (i.c.v.) administrations
or at a volume of 20 µL/mouse for subcutaneous administrations.
2.5.3. Surgery for i.c.v. Injections
For i.c.v. injections, mice were lightly anesthetized with isoflurane, and an incision was made in
the scalp, and the bregma was located. Injections were performed using a 10 µL Hamilton microsyringe
equipped with a 26-gauge needle, 2 mm caudal and 2 mm lateral from the bregma at a depth of 3 mm.
2.5.4. Tail Flick Test
The tail flick latency was obtained using a commercial unit (Ugo Basile, Gemonio, Italy), consisting
of an infrared radiant light source (100 W, 15 V bulb) focused onto a photocell utilizing an aluminum
parabolic mirror. During the trials, the mice were gently hand-restrained using leather gloves. Radiant
heat was focused 3–4 cm from the tip of the tail, and the latency (s) of the tail withdrawal to the thermal
stimulus was recorded. The measurement was interrupted if the latency exceeded the cut off time
(15 s at 15 V). The baseline latency was calculated as mean of three readings recorded before testing
at intervals of 15 min and the time course of latency determined at 15, 30, 45, 60, 90, and 120 min
after treatment. Data were expressed as time course of the maximum percentage effect (%MPE) =
(post-drug latency − baseline latency)/(cut-off time − baseline latency) × 100.
2.5.5. Formalin Test
In the formalin test, the injection of a dilute solution of formalin (1%, 20 µL/paw) into the dorsal
surface of the mouse hind paw evoked biphasic nociceptive behavioral responses, such as licking,
biting the injected paw, or both, occurring from 0 to 10 min after formalin injection (the early phase) and
a prolonged phase, occurring from 15 to 40 min (the late phase). Before the test, mice were individually
placed in a Plexiglas observation cage (30
environment. The total time the animal spent licking or biting its paw during the early and late phase
of formalin-induced nociception was recorded.
×
14
×
12 cm) for one hour, to acclimatize to the testing
2.5.6. Data Analysis and Statistics
Experimental data were expressed as mean
±
s.e.m. In the tail flick test, significant differences
among the groups were evaluated with two-way ANOVA followed by Sidak’s multiple comparisons
test. Formalin test data were analyzed by using one-way ANOVA, followed by Holm–Sidak’s multiple
comparisons test. GraphPad Prism 6.03 software was used for all the analyses. Statistical significance
was set at p < 0.05. The data and statistical analysis comply with the recommendations on experimental
design and analysis in pharmacology.

Biomolecules 2020, 10, 284
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2.6. Stability in Human Plasma Sample Preparation
Five microliters of K6 (500
then incubated at 37
incubation was stopped by adding an equal volume of the sequencing mixture (5% aqueous ZnSO4
solution, MeOH, and ACN; 5:3:2), which precipitated proteins, to achieve a final concentration of
µ
g in 250
µ
L of water) were added to 45
µ
L of fresh human plasma,
±
1 ◦C. Prepared samples were removed at several designated time points and
100
supernatant was directly injected into the HPLC system (Waters model 600 solvent pump and 2996
photodiode array detector, with XBridge BEH 130 C18, 4.6 250 mm, 5 m). The samples were tested
in three independents experiments (n = 3) and reported values represent the mean standard error
µ
g/mL. The mixture was vortexed and centrifuged at 12,000
×
g for 5 min, then 20 µL of clear
×
µ
±
(SEM). Data were analysed using simple regression analysis (significant deviation from zero: F1,13
=
171.9, p < 0.0001; Y = −0.9705 × X + 95.98).
3. Results and Discussion
3.1. Chemistry
Simple modifications of kyna scaffold through the insertion of aromatic substituents [34],
C-terminal derivatization as methyl ester [35], or amide bearing a water-soluble side chain [36
]
have been abundantly documented in the literature, rather than the insertion of kyna into a peptide
sequence. On the contrary, different papers highlight the scarce propensity of the kyn carboxylic
group to react with N-terminus free amino acids involved in the coupling reaction, which renders this
unusual amino acid difficult to manage as a building block for peptide synthesis [22,23].
Taking this in mind, we focused our attention on the functionalization of kyna via a variant of
Steglich esterification with the previously prepared Boc-protected 2-aminoethanol [23] so as to obtain
intermediate
Compound
2
in 60% yield after purification by column chromatography (see General Procedure) [24].
2
was deprotected with a mixture of TFA:DCM = 1:1 at r.t. for 1 h and the so obtained
intermediate was coupled with Boc-N(Me)Phe-OH, following the standard procedure for coupling
reaction [36].
Peptide elongation/deprotection steps were repeated to reach the complete Boc-protected peptide
6
in 66% yield, starting from intermediate compound 5, after silica gel column chromatography
(Scheme 1). The final peptide KA1 has been obtained in 71% yield after RP–HPLC purification.
The purity of the sample was assessed by analytical RP–HPLC recorded at 254 nm and was found to
be ≥95%.

Biomolecules 2020, 10, 284
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Scheme 1. Synthesis of peptide KA1. Reagents and conditions: (a) Boc2O, NaOH 1 M, THF, 10 min at
0 ◦C, then 16 h at r.t. quantitative; (
b
) kynurenic acid, EDC
36 h at r.t., 60% yield after silica gel column chromatography; (
) Boc-N-Me-Phe-OH, EDC
HCl, HOBt, DIPEA, DMF, 10 min at 0 ◦C, then 16 h at r.t., 75% yield after
reaction work-up; ( ) Boc-Gly-OH, EDC
HCl, HOBt, DIPEA, DMF, 10 min at 0 ◦C, then 16 h at r.t., 70%
yield after reaction work-up; ( ) Boc-DAla-OH, EDC
HCl, HOBt, DIPEA, DMF, 10 min at 0 ◦C, then
16 h at r.t., 80% yield after silica gel column chromatography; ( ) Boc-Tyr-OH, EDC HCl, HOBt, DIPEA,
DMF, 10 min at 0 ◦C, then 16 h at r.t., 66% yield from 5, after silica gel column chromatography.
·
HCl, DMAP, DMF, 10 min at 0 ◦C, then
) TFA/DCM 1:1 1 h at r.t. quantitative;
c
(
d
·
e
·
f
·
g
·
Then L-kyn was converted in its Boc-derivative
Tsentalovich et al. 23] to prepare the EM-2 analogs K2 and K3 (Scheme 2). Firstly intermediate
was coupled with H-Phe-NH2, previously prepared following the general procedure of amidation,
to obtain intermediate in 61% yield. The Boc-protecting group was removed from compound
and the so obtained TFA salt was coupled with Boc-Pro-OH to give intermediate in good yield (81%).
7 following the procedure reported by
[
7
8
8,
9
Repeated steps of coupling/purification/deprotection afforded the final compound K2 in 72% yield from
10, and excellent purity after RP–HPLC purification of the crude product. Conversely, Boc-Kynurenine
7
was transformed in the amide Boc-derivative 11 in a 71% yield. Then it was deprotected with a
mixture of TFA:DCM = 1:1 at r.t. for 1 h and the so obtained TFA salt was coupled with BocPhe-OH
to afford intermediate 12 quantitatively. Then repeated steps of coupling/purification/deprotection
were performed to reach peptide K3 in high yield (83% from 14) and excellent purity after RP–HPLC
purification of the crude compound.

Biomolecules 2020, 10, 284
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Scheme 2. Synthesis of peptides K2, K3. Reagents and conditions: (
a
) Boc2O, NaHCO3, NaOH 1 M,
) iBCF, NMM, NH4OH, THF,
10 ◦C, then 16 h at r.t., 71% yield after trituration; (
30 min at c) TFA/DCM 1:1, 1 h at r.t. quantitative;
dioxane/H2O (2:1), 10 min at 0 ◦C, then 30 min at r.t. quantitative; (
b
−
(
(
d
e
) Boc-Phe-OH, EDC.HCl, HOBt, DIPEA, DMF, 10 min at ◦0 ◦C, then 16 h at r.t. quantitative;
) Boc-Pro-OH, EDC HCl, HOBt, DIPEA, DMF, 10 min at 0 C, 16 h at r.t., 72% yield for 13 after
·
trituration, 81% yield for
95% yield for 14 after trituration, 73% yield for 10; (
10 min at 0 ◦C, then 16 h at r.t., 61% yield from 7 after trituration.
9
; (f) Boc-Tyr-OH, EDC
·
HCl, HOBt, DIPEA, DMF, 10 min at 0 ◦C, 16 h at r.t.,
) H-Phe-NH2, EDC HCl, HOBt, DIPEA, DMF,
g
·
Finally, linear peptides K4–K6 have been synthesized as methyl ester, acid, and amide derivatives,
respectively, starting from L-kyn methyl ester 15 (Scheme 3), prepared following the procedure
described in the literature [24,36]. Repeated steps of deprotection/coupling reaction were performed to
reach intermediate 19 in high yield after trituration in Et2O. Boc group removal of intermediate 19
gave K4 in good yield and excellent purity after RP–HPLC purification. Saponification of intermediate
compound 19 afforded 20, following the general procedure; the conversion of the latter in the amide 21
was performed as previously described by Stefanucci et al. [24]. Boc group removal of the linear peptides
20 and 21 afforded products K5 and K6 in good yields (72% and 52%, respectively), and excellent
purity after RP–HPLC purification (98% and 96%, respectively).

Biomolecules 2020, 10, 284
10 of 18
Scheme 3. Synthesis of linear peptides K4–K6. Reagents and conditions: (a) SOCl2, MeOH, 30 min at
0 ◦C, 16 h at r.t., quantitative; (
b
) Boc-Phe-OH, EDC
r.t., 60% after silica gel column chromatography; ( ) TFA/DCM 1:1, 1 h at r.t. quantitative; (
EDC ) Boc-DAla-OH, EDC
HCl, HOBt, DIPEA, DMF, 10 min at 0 ◦C, 16 h at r.t., quantitative; (
HOBt, DIPEA, DMF, 10 min at 0 ◦C, 16 h at r.t., 84% yield after silica gel column chromatography;
·
HCl, HOBt, DIPEA, DMF, 10 min at 0 ◦C, then 16 h at
) Boc-Gly-OH,
HCl,
c
d
·
e
·
(
(
f
g
) Boc-Tyr-OH, EDC
) NaOH 1 M, THF, 3 h at r.t., quantitative; (
·
HCl, HOBt, DIPEA, DMF, 10 min at 0 ◦C, 16 h at r.t., 72% yield after trituration;
) iBCF, NMM, NH4OH (aq), THF, 30 min at
15 ◦C, 2 h at
h
−
r.t., 52% yield after trituration.
3.2. In Vitro Studies
3.2.1. Binding Assays
Kyna and its analog (KYNA1) previously reported by Z
-receptors in vitro 37]. However, after chronic and acute administration, they altered opioid receptor
ádor et al. did not directly bind µ, δ,
κ
[
function in vivo and in vitro through the NMDA receptor co-localized in the cortex and striatum of
mice and rats, though the interaction of opioid receptors and NMDA have been deeply discussed in
the literature [37,38]. Kyna is able to bind to the NMDA receptor at micromolar affinity [38]. To test if
our novel peptides are able to target both of these systems, they were examined in a receptor binding
radioassay using highly specific tritium-labelled primary ligands for opioid and NMDA receptor
binding sites. [3H]DAMGO, [3H]Ile5,6Delt II, and [3H]MK-801 equilibrium competition (displacement)
studies were conducted in rat brain homogenates, while
[3H]HS665 in guinea pig brain homogenates. The novel ligands showed similar equilibrium inhibitory
affinities (Ki value) in the -opioid system as DAMGO except K3 (Table 1, Figure S1A). In the -opioid
system, the ligands showed lower binding affinity (higher Ki) compared to the selective -opioid
κ-opioid receptor tests were performed with
µ
δ
δ

Biomolecules 2020, 10, 284
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receptor selective agonist Ile5,6Delt II (Figure S1B). In the
κ
-opioid receptor system, the compounds
showed higher Ki values than that of the selective κ-opioid agonist HS665 (Figure S1C). In the NMDA
receptor binding assays, the peptides did not produce any competing activity (Figure S1D). Peptide
KA1 possesses the best binding affinity, with a Ki value very close to that of the reference compound
DAMGO (1.08
sequence does not impair its binding potency at the
enkephalin-like structure linked to L-kyn C-terminal amide, is able to bind all three opioid receptors
with significant affinity, showing a moderate preference for the -opioid receptor (affinity ratio 1:18:70
for , k, respectively), whereas compounds K4 and K5 are able to bind only - and -opioid
receptors. It is reasonable to believe that the C-terminal amide derivatization in peptide K6 confers the
ability to bind to the -opioid receptor. Concerning the endomorphin-2 (EM-2) analogs K2 and K3
the replacement of Phe3 with L-kyn improves the binding affinity and selectivity of K2 for
-opioid
receptors with respect to the standard compound EM-2, with a weak affinity for -opioid receptors,
while the incorporation of L-Kyn amide in position 4 causes the loss of selectivity for MOR in favor of
a modest binding affinity for - and -opioid receptors. Peptide K2 shows a Ki value two-folds lower
than that of EM-2 on the -opioid receptor, which let us suppose the positive influence of L-Kyn in
±
0.26 nM vs. 0.90
±
0.28 nM), suggesting that the insertion of kyna into the DAMGO
µ-opioid receptor. The peptide K6, presenting the
µ
µ,
δ
µ
δ
κ
,
µ
κ
µ
δ
µ
position 3 on K2 binding ability.
Table 1. Displacement of [3H]DAMGO, [3H]Ile5,6Delt II, [3H]HS665, and [3H]MK-801 by DAMGO,
Ile5,6Delt II, HS665, MK-801, and oligopeptides in membranes of rat and guinea pig brains. The IC50
values for the MOR, DOR, KOR, and NMDA, according to the competition binding curves (see Figure S1),
were converted into equilibrium inhibitory constant (Ki) values using the Cheng–Prusoff [39] equation.
Ki + S.E.M. (nM) Opioid System
NMDA System
MK-801 a
Ligand
DAMGO a
Ile5,6Delt II a
HS665 b
c
c
c
DAMGO
Ile5,6Delt II
HS665
0.90 ± 0.28
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
c
c
c
n.d.
8.85 ± 0.77
c
c
c
n.d.
n.d.
n.d.
n.d.
2.38 ± 0.25
c
c
c
MK-801
n.d.
11.45 ± 1.04
c
c
EM-2
KYNA
L-kyn
3.16 ± 0.3
n.d.
n.d.
n.d.
n.d.c
n.d.
c
c
c
n.d.
n.d.
n.d.
n.d.
>10000
>10000
c
c
c
KA1
K2
K3
K4
K5
K6
a
1.08 ± 0.26
1.39 ± 0.30
197.3 ± 0.36
2.29 ± 0.28
9.11 ± 0.32
1.84 ± 0.27
554.7 ± 0.8
>10000
1043 ± 0.3
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
158.8 ± 1.6
31.2 ± 0.7
94.4 ± 0.8
32.5 ± 0.8
127.7 ± 0.3
rat brain membrane, b guinea pig brain membrane, c not determined.
3.2.2. Binding-Protein Activation Assays
The effect of kyn or kyna combined peptides on G-protein activation was investigated in
functional [35S]GTP
γ
S binding assays in rat and guinea pig brain membranes. All ligands produced
dose-dependent stimulations described by sigmoid curves (Figure S2). K6 showed higher efficacy
(Emax) than DAMGO (Table 2). Moreover, 10 M cyprodime and 10 M naltrindole, which are selective
MOR and DOR antagonists, respectively [40 41], significantly reversed the agonist effects of the ligands
in rat brain membrane homogenates (Figure S3A). In guinea pig brain membrane homogenates, the
ligands did not activate G-protein except K4 and K6 (Figure S3B). Additionally, 10 M norbinaltorphine
decreased significantly the agonist effect of K4 but did not change the effect of K6 (Table 3). Altogether,
these data reveal that peptide KA1 acts as a selective -opioid agonist, being able to decrease the
GTP S binding percentage under the basal level in the presence of 10 M cyprodime. Peptides K2
and K3 possess a mixed agonist activity profile, while peptides K4–K6 show a modest mixed
µ
µ
,
µ
µ
γ
µ
µ/δ

Biomolecules 2020, 10, 284
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µ/δ-opioid receptor agonism. Probably peptide K6 is the strongest mixed µ/δ/κ opioid agonist due to
its ability to bind protein G with an efficacy value over the basal level in presence of each selective
opioid antagonist at 10 µM.
Table 2. G-protein activation DAMGO, and novel oligopeptides in [35S]GTP
brain membrane homogenates. The values were calculated according to dose-response binding curves.
γS binding assays using rat
Maximal Stimulation (Efficacy)
Emax ± S.E.M. (%)
172.0 ± 3.5
Potency
Log EC50 ± S.E.M.
−6.384 ± 0.101
−6.504 ± 0.076
−7.535 ± 0.354
−6.993 ± 0.422
−6.073 ± 0.172
−5.990 ± 0.111
−5.984 ± 0.054
Ligand
DAMGO
KA1
K2
140.9 ± 1.4
121.6 ± 2.5
K3
114.0 ± 2.1
K4
155.5 ± 4.8
K5
149.2 ± 3.5
K6
209.7 ± 3.4
Table 3. The maximal G-protein efficacy (Emax) of novel oligopeptides in the absence or presence of
the selective MOR antagonist cyprodime and the selective DOR antagonist naltrindole in rat brain
membrane homogenates and in the absence or presence of the selective KOR antagonist norbinaltorphine
in guinea pig brain membrane homogenates in [35S]GTP
according to bar graphs in Figure S3.
γ
S binding assays. The values were calculated
MOR
DOR
Ligand + NTI
KOR
Ligand
Ligand
Ligand + Cyp
Ligand + Nor-BNI
Emax + S.E.M. (%)
102.5 ± 1.3 *
KA1
K2
139.6 ± 8.2
129.9 ± 8.2
118.9 ± 3.0
160.6 ± 5.7
153.1 ± 5.9
211.7 ± 3.1
94.7 ± 3.7 **
88.0 ± 3.3 **
98.5 ± 1.2 **
101.6 ± 2.6 ***
108.8 ± 1.6 **
108.5 ± 2.4 ***
111.7 ± 1.1
92.8 ± 2.0
95.2 ± 2.2
125.2 ± 2.0
106.9 ± 0.3
139.8 ± 2.3
100.7 ± 6.2 ns
100.7 ± 6.0 ns
106.0 ± 6.3 ns
104.9 ± 9.5 *
110.5 ± 5.0 ns
142.1 ± 7.1 ns
88.8 ± 1.9 **
K3
97.8 ± 1.7 **
K4
103.6 ± 3.2 ***
103.8 ± 1.9 ***
113.3 ± 2.9 ***
K5
K6
Experimental data were processed by GraphPad Prism 5.0 using bar graphs. ns: not significant; * p < 0.05; ** p < 0.01
*** p < 0.001 based on unpaired t-tests.
;
3.3. In Vivo Studies
Data obtained from binding experiments indicate K6 as the best of the series; thus, tail flick and
formalin tests were performed in order to evaluate the effects of this novel peptide in two different
pain animal models. The tail flick test measures the time in which the animal withdraws the tail from a
thermal nociceptive stimulus; this time is increased by analgesics. Compounds were centrally injected,
and the response measured from 15 to 120 min after the administration. Compounds K6 and DAMGO
greatly increase the time of response to thermal nociceptive stimuli and both effects were in the same
order to magnitude (Figure 2).
Since it was reported that some opioid peptides such as DAMGO are also peripheral acting [42], we
performed a formalin test administering K6 and DAMGO subcutaneously in mice paws. The formalin
test measures the behavioral response to chemical nociceptive stimuli evoked by a formalin diluted
solution injected in the mice paw. From 0 to 10 min after the injection, an early phase response occurs
with direct stimulation of peripheral nociceptors, while a response to inflammatory pain appears from
15 to 40 min as a late prolonged phase. Compound K6 was able to reduce the nociceptive response to
formalin in the early phase, whereas a light but not significant reduction was induced in the late phase

Biomolecules 2020, 10, 284
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of the formalin test (Figure 3). After the DAMGO injection, we observed a reduction of the nociceptive
behavior both early and late in the formalin test (Figure 3). The formalin early phase, which depends
upon the direct excitement of sensory neurons through TRPA1 cation channel activation [43] of MORs
at the peripheral endings of nociceptors, is responsible for meaningful analgesia [44], and it is not
surprising that K6 and DAMGO reduced formalin-induced nociception in the early phase of the test.
The differences observed in the late phase are probably due to a different metabolic fate of K6 and
DAMGO after subcutaneous administration, depending upon the protease activity that might act in
different ways on K6 and DAMGO chemical structures.
T A IL F L IC K T E S T
1 2 0
1 0 0
****
V
****
****
****
K 6
8 0
6 0
4 0
2 0
0
D A M G O
***
***
9 0
****
****
****
*
****
1 5
3 0
4 5
6 0
1 2 0
T im e (m in ) a fte r tre a tm e n t
Figure 2. The effect of K6 and DAMGO in the tail flick test. Compounds were administered in the
left cerebral ventricle at the dose of 10 g/10 L, and the time to respond to thermal stimuli measured
µ
µ
from 15 to 120 min. V is for vehicle-treated animals. Statistical analysis: two-way ANOVA followed by
Sidak’s multiple comparisons test. * is for p < 0.05, *** is for p < 0.001, and **** is for p < 0.0001 vs. V.
n = 7.
F O R M A L IN T E S T
E A R L Y P H A S E
L A T E P H A S E
2 0 0
1 5 0
1 0 0
5 0
**
***
***
0
V
K 6
T re a tm e n ts
D A M G O
Figure 3. The effect of K6 and DAMGO in the formalin test. Compounds were administered
subcutaneously (s.c.) in the dorsal hind paw of mice at the dose of 100 g/20 L, 15 min before
a s.c. injection of dilute formalin solution (1% in saline, 20 L/paw). Early phase represents the
µ
µ
µ
formalin-induced nociceptive behavior recorded from 0 to 10 min after formalin injection; late phase is
for the formalin-induced nociceptive behavior recorded from 15 to 40 min after formalin injection. V is
for vehicle-treated animals. Statistical analysis: one-way ANOVA followed by Holm–Sidak’s multiple
comparisons test. ** is for p < 0.01 and *** is for p < 0.001 vs. V. n = 7.

Biomolecules 2020, 10, 284
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3.4. Plasma Stability Results
◦
The plasma stability of compound K6 was tested by incubation in human plasma at 37 C.
The degradation curve (Figure 4) was built by plotting the total amount of remaining peptide
(expressed as µg/mL) vs. time (minutes). Concentration data were obtained in triplicate and analyzed
as simple linear regression using GraphPad 8.3.1. The novel compound exhibits good stability in
human plasma, showing a t1/2 = 47 min, according to the results obtained from the tail flick test after
i.c.v. administration.
Figure 4. Stability of compound K6 in human plasma. Concentration of K6 in µg/mL measured from 0
to 60 min in triplicate.
4. Conclusions
Chemical modification of endogenous opioid peptides promotes the development of novel analogs
with increased potency and improved pharmacokinetic properties, e.g., the synthetic bivalent peptide
biphalin enhanced stroke immunohistochemical and behavioral neuroprotection in comparison to
DPDPE and DAMGO, reducing glutamate toxicity and oxidative stress [45–48]. Metabolites of the KP,
especially kyna and L-kyn, play crucial roles in maintaining the normal brain function, preventing
the over-activation of excitatory amino acid receptors, thus offering novel therapeutical opportunities
for brain neuroprotection. In this work, we have synthesized six novel opioid analogs incorporating
the L-kyn and kyna residues at different positions of several opioid peptide scaffolds. They were
characterized by in vitro and in vivo assays to evaluate their ability to bind the NMDA/opioid receptors
and to induce analgesic effects after i.c.v. and s.c. administration. These novel peptides do not
bind to the NMDA receptors, and some of them showed good/high affinity for opioid receptors
with different selectivity profiles. In particular, KA1 exhibits the binding constant (Ki = 1.08) very
close to that of DAMGO for the
µ-opioid receptor and a pronounced selectivity but medium-low
efficacy ( max = 139%); thus the esterification of the ethanolamine portion with kyna does not add any
E
particular advantage to the parent peptide DAMGO. On the other hand, the presence of L-kyn residue
in place of native Phe in position 3 (K2) leads a potent and selective opioid fragment toward MOR
(selectivity ration µ/δ/κ = 1:10,000:750), whereas the substitution in position 4 (K3) leads to a weak and
unselective agonist at MOR and DOR. In the analogs K4–K6, the kyna residue was inserted in the fifth
position of the YaGP peptide, with different C-terminus, respectively, methyl ester, free carboxylic acid,
and amide. K4 and K5 show a similar mixed binding affinity for MOR and DOR, with a preference for
MOR and none or weak affinity for KOR.
On the contrary, peptide K6 shows an interesting behavior since it is able to bind all three
opioid receptors with binding affinity ranging from high to modest ( Kiµ = 1.84 nM, Kiδ = 32.5 nM,
Kiκ = 127.7 nM), with a potency (logEC50
=
−
5.598) and efficacy at MOR (Emax = 211%) higher than
that of DAMGO (Emax = 172%). Its activity on the GTP S binding assay on rat brain membrane
and guinea pig ileum is well antagonized by the co-administration of the selective antagonists for
MOR and DOR at 10 M concentration, prompting us to deeply investigate its anti-nociceptive effect
in vivo. The formalin test, which is a model of inflammatory pain, revealed that the antinociceptive
γ
µ

Biomolecules 2020, 10, 284
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effect exerted by K6 after subcutaneous administration is significant only in the early phase, whereas
DAMGO is also active in the late phase. In the tail flick test, the K6 analgesic profile after i.c.v.
administration is superimposable to that of DAMGO, according to a good stability profile in human
plasma. These data are encouraging to further develop opioid peptides containing kynurenine moieties
since the insertion of kyna and kyn in our opioid model improved, in some cases, the binding affinity
and was able to modulate the selectivity. Also, the possible role played by the metabolism of these
peptides and their possible implication in different neuropathic and chronic pain models is unknown
and worth further investigation.
Supplementary Materials: The following are available online at http://www.mdpi.com/2218-273X/10/2/284/s1,
Figure S1: MOR (A), DOR (B), KOR (C) and NMDA (D) binding affinity of the novel oligopeptides, Figure S2:
The effect of oligopeptides on G-protein activity compared to DAMGO in [35S]GTP
γ
S binding assay in rat brain
membrane homogenates, Figure S3: The effect of novel oligopeptides on G-protein activity in [35S]GTP
γ
S binding
assays in the absence or presence of the selective MOR antagonist cyprodime (Cyp) and the selective DOR
antagonist naltrindole (NTI) in rat brain membrane homogenates (Figure A) and the selective KOR antagonist
norbinaltorphine (nor-BNI) in guinea pig brain membrane homogenates (Figure B).
Author Contributions: Conceptualization, A.S. and S.B.; Data curation, E.S., F.Z., S.P. and G.Z.; Formal analysis,
F.Z. and L.V.; Investigation, A.S., M.N. and S.P.; Methodology, E.S. and M.P.D.; Validation, S.B.; Writing–original
draft, A.S.; Writing–review & editing, A.M., L.V. and S.B. All authors have read and agreed to the published
version of the manuscript.
Funding: This research was supported by the project GINOP 2.3.2-15-2016-00034, provided by National Research,
Development and Innovation Office (NKFI), Budapest, Hungary, and the Ministry of Human Capacities, Hungary
grant 20391-3/2018/FEKUSTRAT.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
KP
kynurenine pathway
kyn
kynurenine
kyna
kynurenic acid
FDA
CNS
Food and Drug Administration
central nervous system
BBB
blood brain barrier
GPR35
SVCT2
MOR
DOR
G protein-coupled receptor 35
Sodium-dependent vitamin C transporter 2
µ-opioid receptor; DOR, δ-opioid receptor
δ-opioid receptor
KOR
k-opioid receptor; GPCRs, G protein coupled receptors
G protein coupled receptors
Nuclear magnetic resonance
trifloroacetic acid
GPCRs
NMR
TFA
ACN
RP-HPLC
TMS
acetonitrile
Reverse Phase High performance liquid chromatography
trimethylsilane
ESI
Electrospray ionization
LRMS
HOBt
DMAP
EDC.HCl
EtOAc
THF
Low Resolution Mass Spectroscopy
1-hydroxybenzotriazole
4-Dimethylaminopyridine
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
ethyl acetate
tetrahydrofurane
NMM
N-methylmorpholine

Biomolecules 2020, 10, 284
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iBCF
isobuthylchloroformiate
DMSO
Boc
Ar
dimethylsulfoxide
tert-butyloxycarbonyl
aryl
EM-2
BSA
i.c.v.
endomorphine-2
Bovine serum albumin
intracerebroventricular
cyprodime
Cyp
NTI
naltrindole
Nor-BNI
DPDPE
DIPEA
DMF
DCM
MeOH
TIPS
norbinaltorphine
[D-Pen,D-Pen5]Enkephalin
N,N-Diisopropylethylamine
dimethylformamide
dichloromethane
methanol
triisopropylsilane
EGTA
GDP
GTP
DMSO
DAMGO
IleDelt II
ethylene glycol-bis(2-aminoethylether)-N,N,N0,N0-tetraacetic acid
guanosine 5’-diphosphate
guanosine-5’-triphosphate
dimethylsulfoxide
[D-Ala2, N-MePhe4, Gly-ol]-enkephalin
Ile5,6-deltorphin II
[
35S]GTPγS guanosine-5’-[35S]thiophosphate
NMDA
Tris-HCl
N-methyl-D-aspartate receptor
tris-(hydroxymethyl)-aminomethane hydrochloride
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