Synthesis, Biological Activity, and NMR-Based Structural Studies of Deltorphin i Analogs Modified in Message Domain with a New α,α-Disubstituted Glycines

Article abstract of DOI:10.1111/cbdd.12730

This article describes new deltorphin I analogs in which phenylalanine residues were replaced by the corresponding (R) or (S)-α-benzyl-β-azidoalanine, α-benzyl-β-(1-pyrrolidinyl)alanine, α-benzyl-β-(1-piperidinyl)alanine, and α-benzyl-β-(4-morpholinyl)-al

Full text of DOI:10.1111/cbdd.12730

Chem Biol Drug Des 2016; 87: 824–832
Research Article
Synthesis, Biological Activity, and NMR-Based
Structural Studies of Deltorphin I Analogs Modified
in Message Domain with a New a,a-Disubstituted
Glycines†
Anika Lasota1, Oliwia Fraczak1, Adriana
table to the presence or absence of a spatial overlap
between the N-terminal message domain and the
C-terminal address domain.
z
Muchowska2, Michał Nowakowski3,
Maciej Maciejczyk4,5, Andrzej Ejchart6 and
Aleksandra Olma1,*
Key words: deltorphin
I analogues, NMR-based studies,
1Institute of Organic Chemistry, Lodz University of
opioid activities, a,a-disubstituted glycines
_
Technology, Zeromskiego 116, 90-924 Lodz, Poland
2Mossakowski Medical Research Centre, Polish Academy
Received 28 July 2015, revised 21 October 2015 and
accepted for publication 16 January 2016
ꢀ
of Sciences, Pawinskiego 5, 01-793 Warsaw, Poland
3Centre of New Technologies, University of Warsaw,
Banacha 2C, 02-097 Warsaw, Poland
4Department of Physics and Biophysics, Faculty of Food
Science, University of Warmia and Mazury,
Opioid peptides include a large group of physiologically
active bioregulators exhibiting a broad spectrum of biologi-
cal activity and interacting with opioid receptors (l, d, j).
Deltorphins are heptapeptides that have been isolated
from the South American frog belonging to the genus
Phyllomedusa (1). Deltorphins show a higher affinity and
selectivity for d-opioid receptors than any other endoge-
nous mammalian compound (2).
Oczapowskiego 4, 10-719 Olsztyn, Poland
5Laboratory of Bioinformatics and Protein Engineering,
International Institute of Molecular and Cell Biology in
Warsaw, ul. Ks. Trojdena 4, 02-109 Warsaw, Poland
6Institute of Biochemistry and Biophysics, Polish Academy
ꢀ
of Sciences, Pawinskiego 5A, 02-106 Warsaw, Poland
*Corresponding author: Aleksandra Olma,
aleksandra.olma@p.lodz.pl
†This article is dedicated to the memory of Andrzej
Lipkowski (deceased November 27, 2014). The peptide
community has lost an excellent scientist and a dear
friend, and he will be missed by all of us who were
fortunate enough to know him and work with him.
Deltorphin I and deltorphin II consist of two parts, a biolog-
ically important N-terminal tripeptide fragment (Tyr-D-Ala-
Phe, the message domain), a binding pharmacophore (3),
and a C-terminal fragment (Asp/Glu-Val-Val-Gly-NH2, the
address domain). Anionic and hydrophobic C-terminal
tetrapeptides decrease l-affinity while at the same time
increasing d-affinity (4). The conformational, topographical,
and stereoelectronic structural features of the opioid pep-
tides are important for interaction with l-, d-, and j-opioid
receptors. Two aromatic amino acids, Tyr1 and Phe3 or
Phe4, are important structural elements because they
interact with opioid receptors. The search for new analogs
of deltorphins is an important direction of study because
they are likely to be effective analgesic agents for the treat-
ment of cancer pain (5) and neuropathic pain (6) with a
low potential for abuse (7,8). Since the discovery of
endogenous amphibian peptides, hundreds of analogs of
the various deltorphins have been synthesized (9–14).
This article describes new deltorphin I analogs in which
phenylalanine residues were replaced by the corre-
sponding (R) or (S)-a-benzyl-b-azidoalanine, a-benzyl-b-
(1-pyrrolidinyl)alanine, a-benzyl-b-(1-piperidinyl)alanine,
and a-benzyl-b-(4-morpholinyl)-alanine residues. The
potency and selectivity of the new analogs were evalu-
ated by a competitive receptor binding assay in the rat
brain using [3H]DAMGO (a l ligand) and [3H]DELT (a d
ligand). The affinity of analogs containing (R) or (S)-a-
benzyl-b-azidoalanine in position
3 to d-receptors
strongly depended on the chirality of the a,a-disubsti-
tuted residue. The conformational behavior of peptides
modified with (R) or (S)-a-benzyl-b-(1-piperidinyl)Ala,
which displays the opposite selectivity, was analyzed
by 1H and 13C NMR. The l-selective Tyr-D-Ala-(R)-
a-benzyl-b-(1-piperidinyl)Ala-Asp-Val-Val-Gly-NH2 lacks
the helical conformation observed in the d-selective Tyr-
D-Ala-(S)-a-benzyl-b-(1-piperidinyl)Ala-Asp-Val-Val-Gly-
NH2. Our results support the proposal that differences
between d- and l-selective opioid peptides are attribu-
It has been proposed that the d-receptor selectivity of del-
torphins is a result of the formation of special non-equal
amphiphilic topography (‘hot-dog’ shape) (3). In such a
conformation, the hydrophilic strain (‘hot-dog’) formed by
ionic and hydrogen bonds between NH2 (Tyr1). . .- COOH
824
ª 2016 John Wiley & Sons A/S. doi: 10.1111/cbdd.12730

Activity and NMR Studies of DT I Analogues
according to procedure described in the literature
(Asp4). . .CONH2 (Gly7) is surrounded by dominating lipo-
philic shells (‘hot-dog roll’). Some very potent and selective
cyclic analogs, which stabilized this conformation, have
supported the proposed model (15). The incorporation
amphiphilic amino acid residues (a-hydroxymethylphenyla-
lanine) also support proposed molecular model of the
active conformation of deltorphins (16).
a
(17,18). All solvents and reagents used for solid-phase
synthesis were of analytical quality and used without fur-
ther purification. Thin layer chromatography (TLC) was per-
formed on UV plates (Fluka Analytical, Silica on TLC
Alufoils, with a 254-nm fluorescent indicator). The coupling
reagents HATU and HOAt were purchased from AK Scien-
tific, Inc. (Union City, USA, CA). All other reagents and sol-
vents were of analytical or HPLC grade and were bought
from Sigma-Aldrich or Avantor Performance Materials
Poland S.A.
The presented paper describes the synthesis and receptor
binding of new analogs in which Phe3 residue in the
deltorphin I sequence was substituted by (R) or (S) a-ben-
zyl-b-azidoalanine, a-benzyl-(1-pyrrolidinyl)alanine, a-ben-
zyl-b-(1-piperidinyl)alanine, and a-benzyl-b-(4-morpholinyl)
alanine (Figure 1). Phenylalanine in position 3 of d-selective
deltorphins and l-selective dermorphin plays a key role
in binding and discrimination between d- and l-opioid
receptors.
Analytical reverse-phase HPLC was performed on
a
GraceSmart C18 column (Grace, 4.6 mm 9 250 mm,
5 lm), flow rate 1.0 mL/min, detection at 220 nm, sol-
vents (A) 0.05% trifluoroacetic acid (TFA) in water, and (B)
0.038% TFA in acetonitrile/water 90:10 in linear gradient
elution. The final peptides were purified by RP-HPLC on a
Thermoseparation Products P400 Spectra System (detec-
tion at 220 nm) using a Gemini C18 column (Phenomenex,
250 mm 9 10 mm, 10 lm), flow rate 3.0 mL/min.
Optically, pure a,a-disubstituted glycines were obtained
from available N-Boc-(R or S)-a-benzylserine b-lactone.
The treatment of N-Boc-(R or S)-a-benzylserine b-lactone
with sodium azide or free heterocyclic amines (pyrrolidine,
piperidine, morpholine) as nucleophile gives suitable, enan-
tiomerically pure N-Boc-(R or S)-a-benzyl-b-azido(sec-
amino)alanines (17,18).
1H NMR spectra of protected amino acids, di- and tripep-
tides were recorded on a Bruker DPX 250 spectrometer
(Bruker Biospin GMBH, Rheinstetten, Germany). Proton
chemical shifts are reported in ppm (d) relative to internal
tetramethylsilane (TMS, d: 0.00). Data are reported as
follows: chemical shift {multiplicity [singlet (s), doublet (d),
triplet (t), quartet (q), and multiplet (m)], coupling constants
[Hz], integration}. ESI-LC-MS was recorded on a Bruker
amaZon speed ETD trap, with an ESI ion source, positive
ion polarity, a maximum resolution mass range, and a
50–2000 m/z range.
The b-azido group is an effective C7-conformation-direct-
ing element, which may be useful for tuning the structures
of other amino acids and polypeptides. However, it has
not been clarified yet whether the azido group can induce
any conformational change via stereoelectronic effects
when introduced into the b-carbon of alanine (19).
Experimental Section
General procedure for synthesis of 2a–2c
Chemistry
To N-Boc-(R or S)-a-benzyl-b-azido(sec-amino)alanine
(1.2 mM) in 3 mL of MeOH, a freshly prepared ethereal
solution of diazomethane was added until the yellow color
of diazomethane persisted. Reaction without stirring was
left overnight. The progress of the reactions was
monitored with TLC (chloroform:methanol 9:1 v/v). Then,
99.5% acetic acid was added carefully to destroy unre-
acted diazomethane and the solvents are removed under
Most chemicals were purchased from Sigma-Aldrich
ꢀ
(Poznan, Poland) and used as received without further
purification. All untreated solvents used were of HPLC
grade. Fluorenylmethyloxycarbonyl (Fmoc)-protected amino
acids and Fmoc-Rink-Amide AM Resin were purchased
from IrisBiotech (Marktredwitz, Germany). (R) and (S) N-
Boc-a-benzyl-b-azido(sec-amino) alanines were obtained
Figure 1: Structure of new deltorphin I
analogs.
Chem Biol Drug Des 2016; 87: 824–832
825

Lasota et al.
vacuum on a rotary evaporator. The crude methyl ester
was diluted with ethyl acetate (25 mL) and washed with
three portions of water, 5% NaHCO3, and brine, dried with
magnesium sulfate, and concentrated under vacuum. N-
Boc-(R or S)-a-benzyl-b-azidoalanines without further
purification were used in the next step, and N-Boc-(R or
S)-a-benzyl-b-(sec-amino)AlaOMe was purified by flash
chromatography (chloroform: methanol 95:5 v/v).
group) or pHꢀ6–7 (analogs containing
a sec-amino
group). Then, aqueous layer was saturated with NaCl and
extracted with ethyl acetate (3 9 15 mL). The combined
ethyl acetate layer was dried over MgSO4 and evaporated
in vacuo. The crude N-protected tripeptides (5a–5d) were
used for the next step.
The structures of all isolated compounds were established
by nuclear magnetic resonance (NMR). Full characteriza-
tion as well as detailed experimental procedures for all
intermediates is available in the online supporting informa-
tion.
General procedure for the deprotection of the
Boc-group
To a solution of N-Boc-(R or S)-a-benzyl-b-azido(sec-
amino)AlaOMe in 1 mL of ethyl acetate, 7 mL of 2 N HCl
in AcOEt was added. After 2 h, next portion of 7 mL HCl
in AcOEt was added and the stirring was continued for 2–
4 h. The reaction was monitored by TLC (chloroform:
methanol 9:1 v/v). After conversion of all starting material,
the product was precipitated with diethyl ether. Precipi-
tated amorphous solids were filtered off and washed with
ethyl ether and used for the next step without further
purification.
General procedure for synthesis of I-VIII
Tetrapeptide resin was prepared by the manual solid-
phase technique on Rink-Amide AM Resin (capacity
0.1 mmol/g), according to standard methods for peptides
synthesized by the Fmoc/tBu strategy. The protected
amino acids were coupled with a threefold excess using
TBTU as a coupling reagent in the presence of HOBt and
DIPEA in DCM. In the case of a positive Kaiser test (20),
the coupling was repeated with a 1.5-fold excess of
reagents. The Fmoc groups were removed by treatment
with 20% piperidine in DMF. The tetrapeptide on resin was
acylated with a twofold excess of N,O-protected tripep-
tides containing (R) or (S)-a-benzyl-b-azido(sec-amino)ala-
nine 5a–5d using HATU as a coupling reagent in the
presence of HOAt and DIPEA in DCM. In the case of a
positive Kaiser test, the coupling was repeated with a 1.2-
fold excess of reagents. The heptapeptides were cleaved
from the resin, and protecting groups were removed
in one step using a mixture of TFA/H2O (95:5 by vol)
(20 mL/100 mg of peptide resin, 3.5 h at room tempera-
ture). The acid solution was concentrated in vacuo, and
the crude peptides were dissolved in water/t-butanol (1:1
by vol), lyophilized, and then purified by RP-HPLC. All hep-
tapeptides were characterized by analytical RP-HPLC and
molecular weight determination.
General procedure for synthesis 3a–3d and 4a–4d
To a stirred solution of Boc-D-alanine or N,O-DiBoc-tyro-
sine (1eq.) in dry DCM, HATU (1eq), HOAt (1 eq) and N-
methylmorpholine (4 eq for monohydrochlorides, or 5 eq
for dihydrochlorides) were added. After 20 min., methyl
ester hydrochloride of amino acid 2a–2d or hydrochloride
of unprotected dipeptide 3a–3d (1 eq) dissolved in 2 mL
of dry dimethylformamide was added. The reaction pro-
gress was controlled by TLC in chloroform: methanol 9:1
v/v. After 20 h, if a significant amount of unreacted sub-
strates were present, additional amount of HATU(0.5 eq)
and HOAt (0.5 eq) and amine (1 eq) was added. Then, the
reaction mixture was stirred at room temperature for 16 h.
The solvent was evaporated under reduced pressure; the
residue was diluted with ethyl acetate and washed with
three portions of water, 1 N NaHSO4 (for dipeptides con-
taining secondary amines, this step was omitted due to
formation of quaternary ammonium salts), 5% NaHCO3,
and brine, dried with magnesium sulfate, and concen-
trated under vacuum. Purification by flash chromatography
(chloroform:methanol 95:5v/v) afforded the desired di- or
tripeptides.
Receptor binding assay
Receptor binding assays were performed as described
previously (16). Crude membrane fractions from rat brain
were prepared as described by Misicka et al. (3) The
radioreceptor binding protocol was based on a protocol
elaborated by Fichna et al. (21) with some modifications.
The modification included different incubation time (60
versus 120 min), bacitracin concentration (30 versus
50 lg/mL), and radioligand choice. The modifications were
implemented to obtain optimal binding conditions. Binding
affinities for l- and d-opioid receptors were determined by
displacing [3H]-DAMGO and [3H]-DELT, respectively, from
adult male Wistar rat brain membrane binding sites.
Binding curves were fitted using nonlinear regression.
Compound potency was expressed as IC50 values
(Table 1).
General procedure for synthesis of 5a–5d
To the solution of 4a–4d (1 mM) in 5 mL of methanol
cooled in an ice bath, 3 mL of 1 N NaOH was added.
Stirring was continued at room temperature until no start-
ing material remained (3–6 h, TLC chloroform: methanol
9:1 v/v). Then, the methanol was evaporated under
reduced pressure at room temperature. The residue was
diluted with 20 mL of water, and the aqueous layer was
washed with diethyl ether (3 9 10 mL) and acidified with
1N NaHSO4 to pHꢀ2–3 (analogs containing an azido
826
Chem Biol Drug Des 2016; 87: 824–832

Activity and NMR Studies of DT I Analogues
Parametrization of modified residues
NMR experiments and computation of peptide
structures
The NMR samples contained 5 mg of Tyr-D-Ala-(S)-a-ben-
zyl-b-(1-piperidinyl)Ala-Asp-Val-Val-Gly-NH2 (V) or 4 mg of
Tyr-D-Ala-(R)-a-benzyl-b-(1-piperidinyl)Ala-Asp-Val-Val-Gly-
NH2 (VI) dissolved in 650 lL of 90:10 H2O/D2O (v/v).
For all natural amino acid residues, standard Amber ff10
force-field parameters were applied (31). The parametriza-
tion of a-benzylo-b-(1-piperydynyl)Ala residue was based
on ff10 parameters for phenylalanine residue. The pipery-
dynyl part of amino acid residue was parameterized in the
following manner. Bonded part of the potential was auto-
matically assigned by antechamber and GAFF force-field
(32,33). Partial charges were determined by fitting them
(RESP) to the electrostatic potential (34) obtained from
quantum mechanical computations at the MP2/6–31G(d,p)
level with Gaussian 03 package (35).
All spectra were measured on an Agilent DDR2 spec-
trometer operating at 600 MHz resonance frequency (1H),
60.8 MHz (15N), and 150.9 MHz (13C) at temperature
25 °C. Temperature calibration was carefully adjusted
using an ethylene glycol reference sample (22). 2D
Homonuclear TOCSY (23) (mixing time 80 ms), ROESY
(24) (mixing time 300 ms), and heteronuclear 1H/15
N
HSQC (25) and 1H/13C HSQC (with the offset, spectral
widths, and 13C–1H coupling constants tuned to either
aliphatic or aromatic carbons) spectra were used to
obtain assignments of the 1H, 15N, and 13C resonances.
Time domain data were acquired using States-TPPI
quadrature detection (26). Water suppression was
achieved with pulsed field gradients echo (27). All chemi-
cal shifts in 1H NMR spectra were reported with respect
to external DSS-d4. Chemical shifts of 13C and 15N sig-
nals were referenced indirectly using the 0.251449530
and 0.101329118 frequency ratios 13C/1H and 15N/1H,
respectively (28). Zero filling and a 90°-shifted squared
sine-bell filter were performed prior to Fourier transforma-
tion. Processed spectra were analyzed with SPARKY soft-
ware (29).
Simulated-annealing procedure
The peptide chain was built with XLEAP program (University
of California, San Francisco, CA, USA) of the Amber pack-
age. To remove bad contacts, 1000 steps of geometry
optimization were applied with steepest-descent energy
minimization method. The chain was heated up from 10 to
1200K in 1 ps molecular dynamics run, followed by 2 ps
of high-temperature dynamics and 12 ps cooling process.
NMR distance restraints were slowly switched on during
first 3 ps of simulated-annealing run. Improper dihedral
restraints on chiral centers were switch on to prevent chi-
rality flipping during the high-temperature dynamics.
Finally, the geometry of the peptide was optimized by
1000 steps of steepest-descent and 2000 steps of conju-
gate-gradient energy minimization procedure. The time
step of the simulation was 1 fs, and generalized Born
solvation model was applied (36–38). The simulated-
annealing cycle was repeated 100 times, and the lowest
energy structure was used as the initial structure for
time-averaged restrained molecular dynamics simulation.
Calibration
Intensities of interproton correlations in ROESY spectra, Iij,
were used in determining appropriate distances rij from the
À6
equation Iij = CÁr
(30). The constant C was calculated
ij
from the intensity of correlation between tyrosine protons
HD and HE of fixed distance assumed to be equal to
MD simulations with time-averaged restraints
The geometry of the initial structure was optimized and
equilibrated in 1 ns MD run with time-averaged restraints
applied. The SHAKE algorithm was used to keep cova-
lent bonds with hydrogens constant and 2 fs time step
ꢁ
2.48 A in case of V. In case of VI, correlation intensities
between tyrosine protons HA and both HB were used
ꢁ
ꢁ
assuming r = 2.5 A for stronger correlation and r = 3 A for
a weaker one.
Table 1: Binding affinities of deltorphin analogs I-VIII to d- and l-opioid receptors
IC50 (nM)
la
IC50 ratio
l/d
Peptide
db
Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2 (DTI) (39)
Tyr-D-Ala-(S)-a-benzyl-b-azidoAla-Asp-Val-Val-Gly-NH2
Tyr-D-Ala-(R)-a-benzyl-b-azidoAla-Asp-Val-Val-Gly-NH2 II
Tyr-D-Ala-(S)-a-benzyl-(1-pyrrolidinyl)Ala-Asp-Val-Val-Gly-NH2 III
Tyr-D-Ala-(R)-a-benzyl-b-(1-pyrrolidinyl)Ala-Asp-Val-Val-Gly-NH2 IV
Tyr-D-Ala-(S)-a-benzyl-b-(1-piperidinyl)Ala-Asp-Val-Val-Gly-NH2
Tyr-D-Ala-(R)-a-benzyl-b-(1-piperidinyl)Ala-Asp-Val-Val-Gly-NH2 VI
Tyr-D-Ala-(S)-a-benzyl-b-(4-morpholinyl)Ala-Asp-Val-Val-Gly-NH2 VII
976 Æ 148
2473 Æ 113
1272 Æ 55.5
1793 Æ 54.7
419 Æ 24.31
2876 Æ 99.5
88 Æ 3.1
3.05 Æ 0.10c
320
3.77
144
0.56
1.11
192
0.13
1.77
1.91
I
655 Æ 108
8.8 Æ 1.0
3178 Æ 430
378.7 Æ 25.1
15.0 Æ 1.2
669 Æ 53.5
2205 Æ 166
1373 Æ 137
V
3907 Æ 231
2624 Æ 116
b
Tyr-D-Ala-(R)-a-benzyl-b-(4-morpholinyl)Ala-Asp-Val-Val-Gly-NH2 VIII
aversus [3H]DAMGO, bversus [3H]DELT, cversus [3H]DPDPE.
Chem Biol Drug Des 2016; 87: 824–832
827

Lasota et al.
was applied. The solvation effects were described by
generalized Born model (36–38). During 20 ns produc-
tion, run proton–proton distance restraints obtained from
NMR experiment were time-averaged over 1 ps time
interval. The average energies of time-averaged distance
restraints were below 1 kcal/mol for both peptides. The
resulting trajectories were clustered with average-linkage
clustering algorithm. The clustering metrics was RMSD
of all heavy atoms of the backbone. The number of
clusters was chosen to minimize Davies–Bourdin index
(DBI) and was equal to seven for Tyr-D-Ala-(S)-a-benzyl-
b-(1-piperidinyl)Ala-Asp-Val-Val-Gly-NH2 (V) and five for
Tyr-D-Ala-(R)-a-benzyl-b-(1-piperidinyl)Ala-Asp-Val-Val-Gly-
NH2 (VI).
(CSPPS) involving the coupling of protected peptide seg-
ments on solid support (the fragment approach). N-
Terminal tripeptides containing a,a-disubstituted glycines
in position 3 were obtained in solution using HATU as a
coupling reagent and then, after deprotection of the car-
boxyl function, were coupled with tetrapeptides on resin.
N,O-Protected tripeptides were obtained by the stepwise
peptide chain elongation in solution. (Scheme 1). The
tetrapeptide Asp-Val-Val-Gly-NH2 was synthesized on
solid phase (SPPS), following standard Fmoc strategy
using TBTU/HOBt for coupling reactions and piperidine
20% solution in DMF for Fmoc group deprotection. The
final heptapeptide resins were obtained by segment con-
densation (fragments 3 + 4). Cleavage from the resin
and removal of the protecting groups were carried out
in one step by treating with a mixture of TFA/H2O (95:5
by vol) (20 mL/100 mg of peptide resin, 3.5 h at room
temperature). The acid solution was concentrated in
vacuo, and the crude peptides were dissolved in water/
t-butanol (1:1 by vol), lyophilized, and then purified by
RP-HPLC.
Results and Discussion
N-Protected (R) and (S) a-benzyl-b-azido(sec-amino)ala-
nines were synthesized from conveniently available b-lac-
tones of N-Boc-(R) and (S)-a-benzylserine by ring opening
with a sodium azide, pyrrolidine, piperidine, or morpholine
as nucleophile. Incorporation of a,a-disubstituted glycines
into peptides in stepwise solid-phase peptide synthesis
(SPPS) is difficult due to their steric hindrance and lower
reactivity. Our attempts to prepare DT I analogs by solid-
phase synthesis using Boc strategy were unsuccessful. The
resulting products, despite the use of reagents for difficult
coupling and prolonged time of reaction, are contaminated
with truncated peptides (penta- and hexapeptides) due to
inefficient coupling of a,a-disubstituted amino acids.
This strategy allows for a full control and monitoring of
the peptide synthesis. The cleavage of heptapeptides
from the resin and the removal of the protecting
groups were performed with TFA:water (95:5 by vol).
All crude analogs were purified to homogeneity by RP-
HPLC, and their structures were verified by mass
spectrometry.
The affinities of deltorphin I analogs for l- and d-receptors
were determined by the radioligand binding assay
described previously (15) using [3H]-DAMGO and [3H]-
DELT as l- and d-receptor-specific ligands, respectively.
The designed peptides I–VII reported here were
obtained by convergent solid-phase peptide synthesis
Scheme 1: SchemeSynthesis of N,O-protected tripeptide units.
828
Chem Biol Drug Des 2016; 87: 824–832

Activity and NMR Studies of DT I Analogues
Table 1 shows the binding affinity of deltorphin I analogs to
d- and l-opioid receptors in comparison with deltorphin I.
and rotating frame, has been the method of choice in
studying conformations of organic and biological mole-
cules (30). Short linear peptides are usually characterized
by high structural flexibility. Therefore, long-range correla-
tions have been seldom observed in their NOESY/ROESY
spectra. Nevertheless, one could expect peptides contain-
ing a,a-disubstituted amino acid residue(s) to exhibit
increased conformational rigidity. Complete assignment of
1H, proton-bearing 13C nuclei was obtained from TOCSY,
Phenylalanine in position 3 plays a key role in general
interaction with receptors of both groups of analogs with
common N-terminal parts, d-selective deltorphins, and
l-selective dermorphin. The incorporation of D-Phe3 in del-
torphins resulted in significant decreases in affinity to d-
receptors and partial decrease of receptor selectivity (40).
Nevertheless, topographical location of the Phe3 phenyl
ring is probably more critical than the chirality of amino
acid in this position. Substitution of Phe3 with very rigid
Aic (tetrahydroisoquinoline-3-carboxylic) or Atc (R as well
as S-2-aminoindan-2-carboxylic acid) increased affinity to
both d- and l-receptors by almost 300 times, whether
replacement Phe3 with a-MePhe decreased affinity to d-
receptors over 100-fold for both (R) and (S)-isomers (41).
Substitution of Phe3 with amphiphilic a,a-disubstituted
glycine ((S)-a-hydroxymethylphenylalanine) gave potent and
a-selective ligand (16). Modification of the critical Phe3 resi-
due to vary steric, electronic, and lipophilic properties may
help to determine what features are important for improv-
ing d and l binding affinities (42).
1
ROESY, and H/13C HSQC spectra.
The representative structures of two dominant clusters of
Tyr-D-Ala-(R)-a-benzyl-b-(1-piperidinyl)Ala-Asp-Val-Val-Gly-
NH2 (VI), with total population over 0.5, are shown in
Figure 2.
The populations of two dominating clusters are nearly
identical. The backbone trace of two structures is very
similar with exception of C-terminus. The C-terminal part
of the peptide can form helix as can be seen in Figure 2
(blue structure). The helical conformation is stabilized
partially by hydrogen bonds, but probably more important
is hydrophobic contact between piperydynyl and Val6, as
can be seen in Figure 3. These contacts seem to drive
helix formation at the C-terminus.
As reported in Table 1, the affinity of analogs containing
(R) or (S)-a-benzyl-b-azidoalanine in position 3 depends on
the Ca chirality of a-benzyl-b-azidoalanine. The replace-
ment of phenylalanine with (R)-a-benzyl-b-azidoalanine
(peptide II) slightly decreases d- and l-receptor affinity in
comparison with parent peptide, whereas the incorpora-
tion of (S) isomer gives analog I, considerably less potent
and d-selective. In analog II, the delocalized charge of azi-
domethyl group in a-benzyl-b-azidoalanine may partially
stabilize the proposed ‘hot-dog’ conformation (3). The
incorporation of more amphiphilic amino acid residues (a-
hydroxymethylphenylalanine) gave better stabilization of
proposed molecular model of the active conformation of
deltorphins (16). The substitution of Phe3 with (R) or (S)-
a-benzyl-(1-pyrrolidinyl)alanine and (R) or (S)-a-benzyl-b-(4-
morpholinyl) results in a loss activity and selectivity (III, IV
and VII, VIII).
The Tyr-D-Ala-(S)-a-benzyl-b-(1-piperidinyl)Ala-Asp-Val-Val-
Gly-NH2 (V) lacks contact which seems to drive helix
formation and the representative structures of the most
populated (0.3) and least populated (0.05) clusters are
shown in Figure 4. These two clusters are similar with a
major conformational difference at the C-terminus.
The introduction of the conformationally restricted a-ben-
zyl-b-(1-piperidinyl)alanine (V, VI) in position 3 of deltorphin
I leads to changes in binding affinities to l- and d-opioid
receptors, which are strongly affected by the configuration
at Ca or topographical location of the Phe3 phenyl ring.
The (S) isomer slightly decreases affinity to d-receptors
and significantly to l-receptors, yielding d-selective ligand.
Changing the configuration of a-benzyl-b-(1-piperidinyl)ala-
nine reverses selectivity as compared to deltorphin I, giving
Tyr-D-Ala-(R)-a-benzyl-b-(1-piperidinyl)Ala-Asp-Val-Val-Gly-
NH2 (VI), the l-selective ligand. In a binding assay, analog
V displays a 192-fold higher selectivity for d-receptor, while
analog VI shows a 7.6-fold higher selectivity for l-recep-
tors (over d-receptors). An NMR study was carried out to
explain the opposite selectivities of analogs V and VI. The
nuclear Overhauser effect (NOE), both in the laboratory
Figure 2: Representative structures of two most populated
clusters of the Tyr-D-Ala-(R)-a-benzyl-b-(1-piperidinyl)Ala-Asp-Val-
Val-Gly-NH2 (VI). Pink and blue structures have populations 0.269
and 0.264, respectively. The C-terminus of blue structure forms
helix stabilized by interaction of piperidinyl with Val-6.
Chem Biol Drug Des 2016; 87: 824–832
829

Lasota et al.
observed, which suggested that the extended conformation
was prevalent.
In conclusion, the binding assay showed that the
replacement of phenylalanine with a-benzyl-b-azidoalanine,
a-benzyl-b-(1-pyrrolidinyl)alanine, a-benzyl-b-(1-piperidinyl)
alanine, and a-benzyl-b-(4-morpholinyl)alanine has a strong
effect on binding affinity. Our result supports the proposal
(45) that differences between d- and l-selective opioid
peptides are attributable to the presence or absence of a
spatial overlap between the N-terminal message domain
and C-terminal address domain.
Acknowledgment
This research was supported by The National Science
Centre (NCN, Poland), grant Preludium 2 (2011/03/N/ST5/
04701, A.L.) and National Science Centre for support with
Sonata Bis 2 Grant No. DEC-2012/07/E/ST4/01386 (M.N.).
NMR measurements were carried out at the Biological and
Chemical Research Centre, University of Warsaw, estab-
lished within the project co-financed by European Union
from the European Regional Development Fund under the
Operational Programme Innovative Economy, 2007 – 2013.
Quantum chemistry and molecular dynamics computations
Figure 3: Hydrophobic contact formed by piperidinyl and Val6
residue in Tyr-D-Ala-(R)-a-benzyl-b-(1-piperidinyl)Ala-Asp-Val-Val-
Gly-NH2 (VI).
ꢀ
were performed at CI TASK in Gdansk.
Conflict of Interest
The authors declare that they have no conflict of interest.
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Appendix S1. Synthesis, biological activity and NMR-
based structural studies of deltorphin I analogues modified
in message domain with a new ,-disubstituted glycines.
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