Synthesis, binding affinities and metabolic stability of dimeric dermorphin analogs modified with β3-homo-amino acids

Article abstract of DOI:10.1002/psc.2869

In this study, proteinogenic amino acids residues of dimeric dermorphin pentapeptides were replaced by the corresponding β³-homo-amino acids. The potency and selectivity of hybrid α/β dimeric dermorphin pentapeptides were evaluated by competeti

Full text of DOI:10.1002/psc.2869

Research Article
Received: 3 December 2015
Revised: 9 February 2016
Accepted: 9 February 2016
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/psc.2869
Synthesis, binding affinities and metabolic
stability of dimeric dermorphin analogs
3
modified with β -homo-amino acids
Oliwia Frączak,a Anika Lasota,a Dagmara Tymecka,b Piotr Kosson,c
Adriana Muchowska,c Aleksandra Misickab,c and Aleksandra Olmaa*
In this study, proteinogenic amino acids residues of dimeric dermorphin pentapeptides were replaced by the corresponding β3-
homo-amino acids. The potency and selectivity of hybrid α/β dimeric dermorphin pentapeptides were evaluated by competetive
receptor binding assay in the rat brain using [3H]DAMGO (a μ ligand) and [3H]DELT (a δ ligand). Tha analog containing β3-homo-
Tyr in place of Tyr (Tyr-D-Ala-Phe-Gly-β3-homo-Tyr-NH-)2 showed good μ receptor affinity and selectivity (IC50 = 0.302, IC50 ratio
μ/δ = 68) and enzymatic stability in human plasma. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.
Keywords: β3-homo-amino acids; hybrid α/β dimeric dermorphin pentapeptides; opioid receptors binding affinity; enzymatic stability in
human plasma
the sequence Tyr-D-AA2-Phe-AA4 (where AA2 and AA4 represent a
certain amino acid), have been reported to show a potent agonist
Introduction
Opioids have been used for the treatment of moderate-to-severe
chronic pain. Unfortunately, they have a large number of side
effects [1,2]. Three main types of opioid receptors, μ (MOR), δ
(DOR) and κ (KOR), are known [3]. The main target for analgesia is
the MOR. Opioid receptors can form homodimers and the following
heterodimers: DOR-KOR, DOR-MOR and KOR-MOR [4–7]. Specifi-
cally, designed ligands that are able to penetrate the BBB are used
to study physiological consequences of opioid receptor homo-
dimerization and heterodimerization, and as new analgesics.
Biphalin is one of the most successful applications of the dimeric
ligand approach to design enkephalin analogs [8]. It is composed
of two tetrapeptide pharmacophores (Tyr-D-Ala-Gly-Phe) con-
nected ‘tail to tail’ by a hydrazine bridge. Biphalin is 257-fold and
6.7-fold more potent than, respectively, morphine and etorphine
(a reference μ agonist), in eliciting antinociception when adminis-
trated with intracerebroventriculary [9], and leads to lesser physical
dependence than morphine [10].
Dermorphin, linear heptapeptide (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2),
has been isolated from skins of the South American frogs
Phylomedusa sauvagei [11]. It shows remarkably high μ selectivity
and an extremely potent, long-acting antinociceptive effect [12]
and has 100-fold higher affinity than morphine for μ opioid recep-
tors [13]. Dermorphin shows potent antinociceptive effects with
peripheral injection routes, such as s.c. and i.v. injections [14].
Because pain relief is mediated mainly through MOR, it is important
to understand the interactions between MOR ligands and the recep-
tor. Since the discovery of dermorphin, more than 100 dermorphin
analogs have been synthesized, and their structure-activity relation-
ships have been investigated [15].
activity for the μ opioid receptor [17], for example, DALDA (D-
AA2 = D-Arg, AA4 = Lys-NH2) [18], TAPA (D-AA2 = D-Arg, AA4 = βAla-
OH) [19] or amidino-TAPA [20]. Dimeric dermorphin analogs were
published by Lazarus et al. [21]. Several dimeric dermorphin
tripeptides, tetrapeptides and pentapeptides connected ‘tail-to-tail’
by a different length bridge were synthesized. The most active in
this series dimeric dermorphin pentapeptide with a hydrazine
bridge (Tyr-D-Ala-Phe-Gly-Tyr-NH-)2 exhibits higher μ affinities than
either dermorphin or DAGO [22].
In this paper, we describe the synthesis, binding affinity and enzy-
matic stability of analogs dimeric dermorphin pentapeptides con-
taining β3-homo-amino acids. The synthesis of α/β hybrid peptides,
modified with homologated proteinogenic amino acids, is a useful
tool in drug design. The substitution of α-amino acids with their β-
isomers in biologically active peptides may result in increased en-
zyme stability [23] and also in a strong influence on peptide confor-
mation [24]. Hybrid α/β peptides fold the backbone in a manner
more similar to the natural α-peptides than full β-peptides, and this,
together with their high intrinsic metabolic stability, makes them
good candidates for drug design [25]. Various β3-homo-amino acids
have been used in the design of several classes of ligands, including
*
Correspondence to: Aleksandra Olma, Institute of Organic Chemistry, Lodz University
of Technology, Zeromskiego 116, 90-924 Lodz, Poland. E-mail: aleksandra.olma@p.
lodz.pl
a Institute of Organic Chemistry, Lodz University of Technology, Zeromskiego 116,
90-924, Lodz, Poland
b Faculty of Chemistry, University of Warsaw, Pasteura 1, 02-093, Warsaw, Poland
The N-terminal tetrapeptide of dermorphin is known to be the
minimum sequence required for opioid activity [16]. Synthetic
peptides derived from the dermorphin tetrapeptide, which has
c
Mossakowski Medical Research Centre, Polish Academy of Sciences, Pawinskiego
5, 01-793, Warsaw, Poland
J. Pept. Sci. 2016; 22: 222–227

Dimeric Dermorphin Analogs Modified with β3-homo-amino Acids
opioid peptides [25–30]. Incorporation of β3-homo-phenylalanin or
its analogs at the 4,4’ positions of biphalin resulted in good opioid re-
ceptor affinities and antinociceptive activity [25,26].
HCl × H-β3-homo-Phe-Gly-OBzl
The general procedure for Boc group removal was followed using
1.3 mM of Boc-β3-homo-Phe-Gly-OBzl, yield= 0.47 g (100%); color-
less oil.
Materials and Methods
Boc-D-Ala-β3-homo-Phe-Gly-OBzl
General
The general procedure for synthesis of protected peptides was
followed using 1.3mM Boc-D-Ala-OH and HCl × H-β3-homo-Phe-
Gly-OBzl; yield = 0.57 g (88%); m.p. = 130–132 °C; Rf[CHCl3:CH3OH
9:1] = 0.69; 1H NMR (CDCl3, 250 MHz) δ (ppm): 1.31 (d, 3H, CH3,
J = 6.85Hz); 1.52 (s, 9H, Boc); 2.40–2.68 (m, 2H,CH2); 2.95–3.06 (m,
2H, CH2); 3.83–3.97 (m, 2H, CH2); 4.47–4.66 (m, 2H, 2 × CH); 5.32 (s,
2H, CH2); 7.25–7.40 (m, 5H, Ar); 7.47–7.51 (m, 5H, Ar).
All solvents and reagents were obtained from commercial suppliers
and used without further purification. All the amino acid derivatives
were purchased from Sigma-Aldrich, except BocTyr(Boc), which
was obtained as described by Shevchenko et al. [31]. Optically pure
isomeric Boc-β3-homo-amino acids were prepared in two-step
Arndt–Eistert homologation of N-protected amino acids [32–34].
General procedure for synthesis of protected dipeptides, tripeptides
and tetrapeptides
HCl × H-D-Ala-β3-homo-Phe-Gly-OBzl
The general procedure for Boc group removal was followed using
1.1 mM of Boc-D-Ala-β3-homo-Phe-Gly-OBzl; yield= 0.47g (99%);
colorless oil.
To a stirred solution of Boc-amino acids (1 mM) in DCM, TBTU (1 mM),
HOBt (1 mM) and DIPEA (3 mM) were added. After stirring the mix-
ture for 10 min, amino component (1 mM) was added. Then the reac-
tion mixture was stirred at room temperature for 24 h. The solvent
was evaporated under reduced pressure; the residue was diluted
with ethyl acetate (20 ml) and washed with 1 N NaHSO4 (three times,
5 ml each), 5% NaHCO3 (three times, 5 ml each) and 5 ml of brine,
dried with magnesium sulfate and concentrated under vacuum.
The crude peptides were purified by flash chromatography or
crystallization.
Boc-Tyr(Boc)-D-Ala-β3-homo-Phe-Gly-OBzl
The general procedure for synthesis of protected peptides was
followed using 1.05 mM Boc-Tyr(Boc)-OH and HCl × H-D-Ala-β3-
homo-Phe-Gly-OBzl; yield= 0.70 g (88%); colorless oil; Rf[CHCl3:
1
CH3OH 9:1] = 0.64; H NMR (CDCl3, 250MHz) δ (ppm): 1.12 (d, 3H,
CH3, J = 7.1 Hz); 1.41 (s, 9H, Boc); 1.53 (s, 9H, Boc); 2.26–2.47 (m,
2H, CH2); 2.88–3.11 (m, 4H, 2 × CH2); 3.85–3.94 (m, 2H, CH2); 4.26–
4.45 (m, 3H, 3 × CH); 5.16 (s, 2H, CH2); 7.06–7.37 (m, 14H, Ar).
General procedure for Boc group removal
Boc peptides were deprotected by 2 N HCl in ethyl acetate or by
90% TFAaq at room temperature for 2 h. Then, ethyl ether was
added to the reaction mixture. Precipitated crystals were filtered
off, washed with diethyl ether and used for the next step without
further purification.
Boc-Tyr(Boc)-D-Ala-β3-homo-Phe-Gly-OH 1a
The general procedure for benzyl ester removal was followed using
0.9mM of Boc-Tyr(Boc)-D-Ala-β3-homo-Phe-Gly-OBzl; yield= 0.53 g
(88%); oil; Rf[CHCl3:CH3OH:CH3CO2H 90:10:2] = 0.57; 1H NMR (CDCl3,
250 MHz) δ (ppm): 1.10 (bs, 3H, CH3); 1.38 (s, 9H, Boc); 1.55 (s, 9H,
Boc); 2.22–2.43 (m, 2H, CH2); 2.88–3.17 (m, 4H, 2 × CH2); 3.91–3.96
(m, 2H, CH2); 4.37–4.58 (m, 3H, 3 × CH); 7.06–7.28 (m, 9H, Ar).
General procedure for methyl ester removal
To a stirred solution of methyl ester (1 mM) in MeOH (1.5 ml), 1 N
NaOHaq (1.5 ml) was added. The reaction mixture was stirred at room
temperature for 2 h. The solvent was evaporated under reduced
pressure at room temperature. The residue was diluted with water
and washed with diethyl ether (three times, 5 ml each), acidified with
1N NaHSO4 and extracted with ethyl acetate (three times, 5 ml each).
The organic layer was dried with magnesium sulfate, and concentrated
under vacuum and used for the next step without further purification.
Boc-Tyr(Bzl)-D-Ala-Phe-βAla-OH 1b
Boc-Phe-βAla-OMe The general procedure for synthesis of
protected peptides was followed using 2.15 mM Boc-Phe-OH and
HCl × H-βAla-OMe; yield = 0.73 g (97%); m.p. = 79–81 °C (m.p. = 84–
1
85 °C [35]); Rf[CHCl3:CH3OH 9:1] = 0.67; H NMR (CDCl3, 250MHz) δ
(ppm): 1.41 (s, 9H, Boc); 2.31–2.48 (m, 2H, CH2); 2.94–3.12 (m, 2H,
CH2); 3.33–3.55 (m, 2H, CH2); 3.64 (s, 3H,CH3); 4.22–4.31 (m, 1H, CH);
7.17–7.33 (m, 5H, Ar).
General procedure for benzyl group removal
To a stirred solution of Bzl-protected peptides (1 mM) in AcOH
(5 ml), Pd/Cact in MeOH was added. The reaction mixture was stirred
under a hydrogen atmosphere at room temperature for 4 h. The
catalyst was filtered off through Celite, and the solvent was evapo-
rated under reduced pressure. Purification by crystallization or flash
chromatography, as needed, afforded the desired peptides.
HCl × H-Phe-βAla-OMe
The general procedure for Boc group removal was followed using
2.1 mM of Boc-Phe-βAla-OMe; yield = 0.59 g; (98%); m.p. = 146–147 °C.
Boc-Tyr(Boc)-D-Ala-β3-homo-Phe-Gly-OH 1a
Boc-D-Ala-Phe-βAla-OMe
Boc-β3-homo-Phe-Gly-OBzl The general procedure for synthesis of
protected peptides was followed using 1.5mM of Boc-β3-homo-
Phe-OH and HCl × H-Gly-OBzl; yield= 0.57g (89%); m.p. = 127–
130 °C; Rf[CHCl3:CH3OH 9:1] = 0.59; 1H NMR (CDCl3, 250 MHz) δ
(ppm): 1.40 (s, 9H, Boc); 2.31–2.53 (m, 2H, CH2); 2.82–3.01 (m, 2H,
CH2); 4.06–4.16 (m, 3H, CH2, CH); 5.20 (s, 2H, CH2); 7.19–7.38 (m,
10H, Ar).
The general procedure for synthesis of protected peptides was
followed using 2.0 mM of Boc-D-Ala-OH and HCl × H-Phe-βAla-
OMe; yield = 0.80 g (95%); m.p. = 100–102 °C; Rf[CHCl3:CH3OH 9:1]
= 0.70; 1H NMR (CDCl3, 250MHz) δ (ppm): 1.28 (d, 3H, CH3,
J = 7 Hz); 1.44 (s, 9H, Boc); 2.33–2.51 (m, 2H,CH2); 3.01–3.14 (m, 2H,
CH2); 3.39–3.47 (m, 2H, CH2); 3.65 (s, 3H, CH3); 4.01–4.14 (t, 1H, CH,
J = 7 Hz); 4.60 (q, 1H, CH, J = 7 Hz); 7.14–7.33 (m, 5H, Ar).
J. Pept. Sci. 2016; 22: 222–227
wileyonlinelibrary.com/journal/jpepsci

O. FRĄCZAK ET AL.
HCl × H-D-Ala-Phe-βAla-OMe
p. = 143–145 °C (m.p. = 149–150 °C [37]); Rf[CHCl3:CH3OH:CH3CO2H
90:10:2] = 0.53; 1H NMR (CDCl3, 250 MHz) δ (ppm): 1.09 (bs, 3H,
CH3); 1.36 (s, 9H, Boc); 1.54 (s, 9H, Boc); 2.89–3.23 (m, 4H, 2 × CH2);
3.98 (bs, 2H, CH2); 4.56–4.75 (m, 3H, 3 × CH); 7.07–7.23 (m, 9H, Ar).
The general procedure for Boc group removal was followed using
1.8 mM of Boc-D-Ala-Phe-βAla-OMe; yield = 0.64 g (99%); colorless oil.
Boc-Tyr(Bzl)-D-Ala-Phe-βAla-OMe
General procedure for synthesis of diacylated hydrazides
The general procedure for synthesis of protected peptides was
followed using 1.2 mM of Boc-Tyr(Bzl)-OH and HCl × H-D-Ala-Phe-β
Ala-OMe; yield = 0.70 g (87%); colorless oil; Rf[CHCl3:CH3OH 9:1]
= 0.60; 1H NMR (CDCl3, 250 MHz) δ (ppm): 1.11 (d, 3H, CH3,
J = 6.85 Hz); 1.40 (s, 9H, Boc); 2.45 (t, 2H, CH2, J=6.15Hz); 2.92–3.19
(m, 4H, 2 × CH2); 3.28–3.57 (m, 2H, CH2); 3.64 (s, 3H, CH3); 4.23–4.39
(m, 2H, 2 × CH); 4.58 (q, 1H, CH, J = 7.3Hz); 5.04 (s, 2H, CH2);
6.90–7.10 (m, 4H, Ar); 7.17–7.45 (m, 10H, Ar).
To a stirred solution of Boc-amino acid (2.5 mM) in DCM : DMF (1:1),
TBTU (2.5 mM), HOBt (2.5 mM), and DIPEA (7.5 mM) were added. After
stirring the mixture for 10 min, hydrazine dihydrochloride (1 mM) was
added. Then the reaction mixture was stirred at room temperature for
24 h. The solvent was evaporated under reduced pressure; the resi-
due was precipitated out by the addition of ethyl acetate, filtered,
washed with ethyl acetate and distilled water and dried.
(Boc-Tyr(Bzl)-NH-)2 2a
Boc-Tyr(Bzl)-D-Ala-Phe-βAla-OH 1b
The general procedure for synthesis of diacylated hydrazide 2a was
followed using 5 mM of (Boc-Tyr(Bzl)-OH and 2 mM of hydrazine
dihydrochloride; yield = 1.31 g (89%); m.p. = 196–198 °C (m.p. = 210–
212 °C [38]); Rf[CHCl3:CH3OH 95:5] = 0.59; Rf[CHCl3:CH3OH 9:1] = 0.73.
The general procedure for methyl ester removal was followed using
1.0mM of Boc-Tyr(Bzl)-D-Ala-Phe-βAla-OMe; yield = 0.58 g (88%);
colorless oil; Rf[CHCl3:CH3OH:CH3CO2H 90:10:2] = 0.62; 1H NMR
(CDCl3, 250 MHz) δ (ppm): 1.12 (bs, 3H, CH3); 1.37 (s, 9H, Boc);
2.43–2.54 (m, 2H, CH2); 2.89–3.18 (m, 4H, 2 × CH2); 3.34–3.55 (m,
2H, CH2); 4.44–4.61 (m, 2H, 2 × CH); 4.78–4.89 (m, 1H, CH); 5.01 (s,
2H, CH2); 6.87–7.45 (m, 14H, Ar).
(Boc-β3-homo-Tyr(Bzl)-NH-)2 2b
The general procedure for synthesis of diacylated hydrazide 2b was
followed using 5 mM of (Boc-β3-homo-Tyr(Bzl)-OH and 2 mM of
hydrazine dihydrochloride; yield= 1.03 g (67%); m.p. = 217–219 °C;
Rf[CHCl3:CH3OH 95:5] = 0.44; Rf[CHCl3:CH3OH 9:1] = 0.54.
Boc-Tyr(Boc)-D-Ala-Phe-Gly-OH 1c
Boc-Phe-Gly-OBzl The general procedure for synthesis of
protected peptides was followed using 2 mM of Boc-Phe-OH and
HCl × H-Gly-OBzl; yield = 0.82 g (99%); m.p. = 131–132 °C (m.p.
= 132–134 °C [36]); Rf[CHCl3:CH3O 9:1] = 0.52; 1H NMR (CDCl3,
250 MHz) δ (ppm): 1.39 (s, 9H, Boc); 2.98–3.16 (m, 2H, CH2);
3.92–4.13 (m, 2H, CH2); 4.37–4.45 (m, 1H, CH); 5.16 (s, 2H, CH2);
7.18–7.41 (m, 10H, Ar).
(HCl × H-Tyr(Bzl)-NH-)2 3a
The general procedure for Boc group removal was followed using
1.5 mM of (Boc-Tyr(Bzl)-NH-)2; yield= 0.91g (99%); m.p. = 242–
245 °C.
(HCl × H-β3-homo-Tyr(Bzl)-NH-)2 3b
The general procedure for Boc group removal was followed using
1.5 mM of (Boc- β3-homo-Tyr(Bzl)-NH-)2; yield= 0.95 g (99%); m.p.
= 265–268 °C.
HCl×H-Phe-Gly-OBzl
The general procedure for Boc group removal was followed using
1.9mM of Boc-Phe-Gly-OBzl; yield = 0.64 g (97%); m.p. = 126–128 °C.
General procedure for synthesis of unprotected decapeptides I–VI
Boc-D-Ala-Phe-Gly-OBzl
To a stirred solution of N,O-protected pentapeptides 1a–1c (2.5 eq.)
in DMF, HATU (2.5 eq.) and DIPEA (5 eq.) were added. After stirring
the mixture for 10 min, diacylated hydrazides 3a or 3b (1 eq.) was
added. Then the reaction mixture was stirred at room temperature
for 48 h. The solvent was evaporated under reduced pressure; the
residue was diluted with ethyl acetate and washed with three por-
tions of 1 N NaHSO4, 5% NaHCO3 and brine, dried with magnesium
sulfate and concentrated under vacuum. The crude-protected
decapeptides were purified by flash chromatography and then were
deprotected using general procedures for Boc and benzyl groups re-
moval. Unprotected decapeptides I–VI were purified by RP-high-
performance liquid chromatography (HPLC). The purity of the final
TFA salts was assessed by analytical HPLC and ESI-MS (Table 1).
The general procedure for synthesis of protected peptides was
followed using 1.5 mM of Boc-D-Ala-OH and HCl×H-Phe-Gly-OBzl;
yield = 0.70 g (97%); m.p. = 158–160 °C; Rf[CHCl3:CH3O 9:1] = 0.64;
1H NMR (CDCl3, 250 MHz) δ (ppm): 1.22 (d, 3H, CH3, J = 7 Hz); 1.40
(s, 9H, Boc); 3.04–3.21 (m, 2H, CH2); 3.84–4.16 (m, 3H, CH2, CH);
4.69 (q, 1H, CH, J = 7 Hz); 5.15 (s, 2H, CH2); 7.17–7.37 (m, 10H, Ar).
HCl × H-D-Ala-Phe-Gly-OBzl
The general procedure for Boc group removal was followed using
1.45 mM of Boc-D-Ala-Phe-Gly-OBzl; yield = 0.60 g (98%); m.p.
= 113–115 °C.
Boc-Tyr(Boc)-D-Ala-Phe-Gly-OBzl
The general procedure for synthesis of protected peptides was
followed using 1.4 mM of Boc-Tyr(Boc)-OH and HCl × H-D-Ala-Phe-
Gly-OBzl; yield = 0.95 g (91%); oil; Rf[CHCl3:CH3O 9:1]=0.62; 1H NMR
(CDCl3, 250 MHz) δ (ppm): 1.26 (bs, 3H, CH3); 1.37 (s, 9H, Boc); 1.54
(s, 9H, Boc); 2.87–3.10 (m, 4H, 2 × CH2); 3.99–4.33 (m, 4H, CH2,
2 × CH); 4.63–4.75 (m, 1H, CH); 5.15 (s, 2H, CH2); 7.05–7.36 (m, 14H, Ar).
Results
Optically pure N-Boc-β3-homo-tyrosine(Bzl) and N-Boc-β3-homo-
phenylalanine were prepared in two-step Arndt–Eistert homologa-
tion of protected amino acids [32–34]. Dipeptides, tripeptides and
tetrapeptides were synthesized by conventional method in solution
according to Scheme 1.
The final step of the synthesis included acylation of the amino
groups in the symmetric dihydrazides 3a or 3b by N,O-protected
tetrapeptides 1a–1c (Scheme 2).
Boc-Tyr(Boc)-D-Ala-Phe-Gly-OH 1c
The general procedure for benzyl ester removal was followed using
1.3mM of Boc-Tyr(Boc)-D-Ala-Phe-Gly-OBzl; yield= 0.73 g (86%); m.
wileyonlinelibrary.com/journal/jpepsci
J. Pept. Sci. 2016; 22: 222–227

Dimeric Dermorphin Analogs Modified with β3-homo-amino Acids
Table 1. Structures and the physicochemical properties of the dimeric pentapeptide dermorphin analogs I–VI
Peptides
MW
m/z [M + H]+
RP-HPLC
tR (min)a
Formula
Calc.
Found
Purity
(Tyr-D-Ala-Phe-Gly-Tyr-NH-)2 I
C64H74N12O14
C66H78N12O14
C66H78N12O14
C66H78N12O14
C68H82N12O14
C68H82N12O14
1235.3
1263.4
1263.4
1263.4
1291.5
1291.5
1236.5
1264.5
1264.5
1264.5
1292.5
1292.5
14.12
14.27
13.82
13.99
13.63
13.71
98
96
97
99
98
97
(Tyr-D-Ala-β3-homo-Phe-Gly-Tyr-NH-)2 II
(Tyr-D-Ala-Phe-βAla-Tyr-NH-)2 III
(Tyr-D-Ala-Phe-Gly-β3-homo-Tyr-NH-)2 IV
(Tyr-D-Ala-β3-homo-Phe-Gly-β3-homo-Tyr-NH-)2 V
(Tyr-D-Ala-Phe-βAla-β3-homo-Tyr-NH-)2 VI
aLinear gradient 10–60% B, 20 min, 20 min flow rate 3 ml/min [solvents (A) 0.05% TFA in water and (B) 0.038% TFA in acetonitrile/H2O, 90:10].
Table 2. Binding affinities of the dimeric pentapeptide dermorphin an-
alogs I–VI to δ and μ opioid receptors
Peptides
IC50 [nM]
μ/δ
μ
δ
Dermorphin
1.22 0.13a
0.243 0.005
0.27 0.04a
18.20 0.43
178.6 18a
8.754 0.26
7.88 0.07a
203.7 9.4
146.4a
36.0
(Tyr-D-Ala-Phe-
Gly-Tyr-NH-)2 I
29.2a
(Tyr-D-Ala-β3-homo-Phe-
Gly-Tyr-NH-)2 II
11.2
(Tyr-D-Ala-Phe-βAla-
Tyr-NH-)2 III
3.543 0.169
0.302 0.006
11.2 0.68
85.34 1.83
20.55 1.22
>10000
24.1
68.0
—
(Tyr-D-Ala-Phe-Gly-β3-
homo-Tyr-NH-)2 IV
(Tyr-D-Ala-β3-homo-
Phe-Gly-β3-homo-
Tyr-NH-)2 V
Scheme 1. Synthesis of protected tetrapeptides 1a–1c.
(Tyr-D-Ala-Phe-βAla-β3-
homo-Tyr-NH-)2 VI
40.7 2.9
645 45.8
15.8
The crude peptides were purified by RP-HPLC. The purity of the
final TFA salts was assessed by analytical HPLC (purity >96–99%)
and ESI-MS (Table 1).
a[20]
Receptor binding assay
Receptor binding assays were performed as described previously
[39]. Rat membrane preparation followed the procedure described
by Misicka et al. [40]. The radioreceptor-binding protocol was based
on a study elaborated by Fichna et al. [41] with some modifications.
The modification included different incubation times (60 min vs
120 min), bacitracin concentrations (30μg/ml vs 50 μg/ml) and
radioligand choices. The modifications were implemented in order
to obtain optimal binding conditions. Binding affinities for μ and δ
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 2).
Scheme 2. Synthesis of dimeric dermorphin pentapeptides analogs I–VI.
J. Pept. Sci. 2016; 22: 222–227
wileyonlinelibrary.com/journal/jpepsci

O. FRĄCZAK ET AL.
Figure 1. Stability of dimeric pentapeptide dermorphin analogs I and IV in human plasma in vitro.
Plasma stability studies of I and IV
with dermorphin yet maintain selectivity for μ receptor. The
replacement of tyrosine at positions 5 and 5′ with β3-homo-tyrosine
(IV) exhibits no significant difference relative to I in its affinity for
the μ receptor, and lower affinity for the δ receptor than I giving
the most active and μ selective analog containing β3-homo-amino
acid residue in this series. Similar results were obtained by the incor-
poration of β3-homo-amino acids in positions 4 and 4′ of biphalin.
The binding affinity and δ selectivity have been improved or
remained unchanged. Biphalin analog modified with β3-homo-
Phe in position 4,4′ showed remarkable binding affinity, with
Kiδ = 0.72 and Kiμ = 1.1nM and an increased enzymatic stability in
human plasma [25]. The incorporation of β3-homo-Phe(NO2) in
position 4,4′ increases μ-affinities and slightly decreases δ-affinities
(IC50μ = 0.72 nM; IC50δ = 4.66 nM [43]). It confirms that homologa-
tion of residue connected with linker in dimeric opioid analogs is
generally well tolerated and improves activity and selectivity.
The enzymatic stability of peptide drugs is important if the drug
candidate is to be considered for therapeutic use. The resistance of
the I and IV was determined by HPLC analysis of about 0.5μM
samples incubated with human plasma. Analog I exhibited good
stability to human plasma, whilst analog IV, containing β3-homo-ty-
rosine in position 5,5′, was found to be extremely stable in human
plasma. It was resistant to the blood protease degradation for
96 h (Figure 1).
Plasma stability studies were performed as described previously
[42], with some modifications. The stability of the peptides I and
IV was tested in human plasma obtained from a healthy donor. A
stock solution of the peptide was prepared by dissolving 0.44 μM
(I) or 0.27 μM (IV) in 1 ml of water. In Eppendorf tubes, samples of
human plasma (50 μl) were temperature equilibrated at 37 1 °C
for 5 min before adding 50μl of the peptide stock solution. The
time was recorded at different intervals (0 , 1, 2, 3, 4, 5, 6, 12, 24,
48 and 96 h). Next, ethanol (200 μl) was added to the samples in or-
der to precipitate the plasma proteins. These cloudy mixtures were
shaken 1 min and cooled in a refrigerator (4°C) for 5 min and subse-
quently centrifuged for 10 min at 2000 g (Eppendorf centrifuge).
The reaction supernatant was then analyzed by HPLC/MS using
LCMS-2010EV Shimadzu, a Phenomenex Jupiter 4u Proteo 90A;
C12 (25 cm × 2 mm × 4 μm) column, a non-linear gradient was used:
0–63% B for 40 min followed by an increase to 97% B from 40 to
45 min, at a solvent flow of 0.3 ml/min. Solvent solutions were
solvent A (0.05% TFA in water) and solvent B (0.05% TFA in
acetonitrile). As an internal standard, Z-Val-OH was used. Addition-
ally, the activity of the human plasma was tested using
Endomorphin-2 (0.47μM/ml) as a control sample, as it is known
that Endomorphin-2 is unstable in human plasma.
The metabolic stability of biphalin is lower (the half-life found after
incubation in human plasma was 24 h) (Misicka A, Tymecka D. per-
sonal communication). Present results suggest that modification of
flexibility (an additional CH2 group in the main chain of the peptide)
may facilitate fitting to μ opioid receptor and that these compounds
will be not be degraded enzymatically before having an opportunity
to produce the desired receptor mediated, analgesic effects.
Preliminary data were presented on the 24th American Peptide
Symposium [44].
Discussion
The general strategy for the synthesis of dimeric dermorphin
analogs by (2 × 4) + 2 fragment coupling was similar to the
procedure for synthesis of biphalin, originally described by
Lipkowski et al. [8]. Affinities of α,β-peptides, dimeric dermorphin
analogs, for μ receptor and δ receptor were determined by the
radioligand-labeled binding assay described previously using
[3H]DAMGO and [3H]DELT as
μ receptor-specific and δ
Acknowledgements
receptor-specific ligands, respectively. Table 2 shows the bind-
ing affinity of α,β-peptide analogs of dermorphin to δ opioid
and μ opioid receptors in comparison with dermorphin. Dimeric
dermorphin pentapeptide analog I exhibits five times better af-
finity to μ receptor and 20 times to δ receptor than dermorphin,
and it is comparable with previously described by Lazarus et al.
[21].
This research was supported by the Young Scientists’ Fund no. W-3/
FMN/5G/2014 at the Faculty of Chemistry, Lodz University of Technol-
ogy (Poland) and scholarship no. RNB/WFS/19/2014 from the Internal
Scholarship Fund at the Lodz University of Technology (Poland).
The study was partially carried out at the Biological and Chem-
ical Research Centre, University of Warsaw, established within the
project cofinanced by the EU from the European Regional Develop-
ment Fund under the Operational Programme Innovative Econ-
omy, 2007–2013, and with the use of the CePT infrastructure
financed by the same EU program.
Our studies have shown that analogs containing of β3-homo-
amino acids in positions 3,3′ (II), 4,4′ (III), 5,5′ (IV), 3,3′, 5,5′ (V) and
4,4′, 5,5’(VI) exhibit various degree of affinity to μ and δ receptors.
Analogs II, III, V and VI have better or reduced μ affinity compare
wileyonlinelibrary.com/journal/jpepsci
J. Pept. Sci. 2016; 22: 222–227

Dimeric Dermorphin Analogs Modified with β3-homo-amino Acids
analogues of the opioid peptide biphalin: mixed α/β3-peptides. J. Med.
Chem. 2013; 56: 3419–3423.
References
1 Holzer P. New approaches to the treatment of opioid-induced
constipation. Eur. Rev. Med. Phermacol. Sci. 2008; 12: 119–127.
2 Porreca F, Ossipov MH. Nausea and vomiting side effects with opioid
analgesics during treatment of chronic pain: mechanisms, implications,
and management options. Pain Med. 2009; 10: 654–662.
3 Dhawan B, Cesselin F, Raghubir R, Reisine T, Bradley P, Portoghese P,
Hamon M. International Union of Pharmacology. XII. Classification of
opioid receptors. Pharmacol. Rev. 1996; 48: 567–592.
26 a Frączak O, Lasota A, Kosson P, Leśniak A, Muchowska A, Lipkowski AW,
Olma A. Biphalin analogues containing β3-homo-amino acids at the 4,4’
positions: synthesis and opioid activity profiles. Peptides 2015; 66: 13–18;
bFrączak O, Lasota A, Podwysocka D, Olma A, Leśniak A, Lipkowski AW.
Analogues of biphalin containing β3-homo-amino acids, 21 Polish
Peptide Symposium, 2011, abstract book P15/I http://21pps.umb.edu.
pl/conference_book_suprasl_2011.pdf
27 Podwysocka D, Kosson P, Lipkowski AW, Olma A. TAPP analogues
containing β3-homo-amino acids: synthesis and receptor binding.
J. Pept. Sci. 2012; 18: 556–559.
28 Wilczynska D, Kosson P, Kwasiborska M, Ejchart A, Olma A. Synthesis and
receptor binding of opioid peptide analogues containing β3-homo-
amino acids. J. Pept. Sci. 2009; 15: 777–782.
29 Bozu B, Fulop F, Toth GK, Toth G, Szucs M. Synthesis and opioid binding
activity of dermorphin analogues containing cyclic β-amino acids.
Neuropeptides 1997; 31: 367–372.
30 Cardillo G, Gentilucci L, Qasem AR, Sgarzi F, Spampinato S.
Endomorphin-1 analogues containing β-proline are μ-opioid receptor
agonists and display enhanced enzymatic hydrolysis resistance. J. Med.
Chem. 2002; 45: 2571–2578.
31 Shevchenko VP, Nagaev IY, Alfeeva LY, Andreeva LA, Shevchenko KV,
Myasoedov NF. Synthesis of Tyr-Pro-Phe-Val-Glu-L-[3,4-H]Pro-Ile-Tyr-D-
Ala-Phe-Gly-Tyr-L-[3,4-H]Pro-Ser-NH2, and Tyr-D-Ala-Phe-Gly-Tyr-D-[3,4-
H]Pro-Ser-NH2, labeled analogs of human-casomorphin and
dermorphin. Radiochem. 2004; 46: 67–76.
32 Linder MR, Steurer S, Podlech J. (S)-3-(tert-butyloxycarbonylamino)-4-
phenylbutanoic acid. Org. Synth. 2002; 79: 154–160.
33 Koch K, Podlech J. Exceptionally simple homologation of protected α-
and β-amino acids in the presence of silica gel. Synth. Commun. 2005;
35: 2789–2794.
4 Gomes I, Jordan B, Gupta A, Trapaidze N, Nagy V, Devi L.
Heterodimerization of μ and δ opioid receptors: a role in opiate
synergy. J. Neurosci. 2000; 20: RC110.
5 Portoghese PS, Lunzer M. Identity of the putativey δ1-opioid receptor as a δ–κ
heteromer in the mouse spinal cord. Eur. J. Pharmacol. 2003; 467: 233–234.
6 Wang D, Sun X, Bohn L, Sadee W. Opioid receptor homo- and
heterodimerization in living cells by quantitative bioluminescence
resonance energy transfer. Mol. Pharmacol. 2005; 67: 2173–2184.
7 George SR, Fan T, Xie Z, Tse R, Tam V, Varghese G, O’Dowd B.
Oligomerization of μ- and δ-opioid receptors. J. Biol. Chem. 2000; 275:
26128–25135.
8 Lipkowski AW, Konecka AM, Sroczyńska I. Double-enkephalins-
synthesis, activity on guinea-pig ileum, and analgesic effect.
Peptides 1982; 3: 697–700.
9 Horan PJ, Mattia A, Bilsky EJ, Weber S, Davis TP, Yamamura HI,
Malatynska E, Appleyard SM, Slaninova J, Misicka A, Lipkowski AW,
Hruby VJ, Porecca F. Antinociceptive profile of biphalin, a dimeric
enkephalin. J. Pharmacol. Exp. Ther. 1993; 265: 1446–1454.
10 Yamazaki M, Suzuki T, Narita M, Lipkowski AW. The opioid peptide
analogue biphalin induces less physical dependence than morphine.
Life Sci. 2001; 69: 1023–1028.
11 Montecucchi PC, de Castiglione R, Piani S, Gozzini L, Erspamer V. Amino acid
composition and sequence of dermorphin, a novel opiate-like peptide from
the skin of Phyllomedusa sauvagei. J. Peptide Protein Res. 1981; 17: 275–283.
12 Erspamer V. The opioid peptides of the amphibian skin. Int. J. Dev.
Neurosci. 1992; 10: 3–30.
13 Melchiorri P, Negri L. The dermorphin peptide family. Gen. Pharmacol.
1996; 27: 1099–1107.
14 Puglisi-Allegra S, Castellano C, Filibeck U, Oliverio A, Melchiorri P.
Behavioural data on dermorphins in mice. Eur. J. Pharmacol. 1982; 82:
223–227.
34 Podwysocka D, Kosson P, Lipkowski AW, Olma A. TAPP analogues
containing β3-homo-amino acids: synthesis and receptor binding.
J. Pept. Sci. 2012; 18: 556–559.
35 Iden HS, Lubell WD. 1,4-Diazepinone and pyrrolodiazepinone syntheses
via homoallylic ketones from cascade addition of vinyl grignard reagent
to α-aminoacyl-β-amino esters. Org. Lett. 2006; 8: 3425–3428.
36 Wang SS, Gisin BF, Winter DP, Makofske R, Kulesha ID, Tzougraki C,
Meienhofer J. Facile synthesis of amino acid and peptide esters under
mild conditions via cesium salts. Org. Chem. 1977; 42: 1286–1290.
37 Korshunova GA, Petrova MG, Taskaeva YM, Sumbatyan NV, Shvachkin YP.
Nucleo amino acids and nucleopeptides. XIV. Synthesis and properties of
nucleo amino acid analogs of the 1-5 fragment of dermorphine. J. Gen.
Chem. USSR (Engl. Transl.) 1987; 57: 1246–1252 1393-1401.
15 Mizoguchi H, Bagetta G, Sakurada T, Sakurada S. Dermorphin tetrapeptide
analogs as potent and long-lasting analgesics with pharmacological
profiles distinct from morphine. Peptides 2011; 32: 421–427.
16 Broccardo M, Erspamer V, Erspamer GF, Improta G, Linari G, Melchiorri P,
Montecucchi PC. Pharmacological data on dermorphins, a new class of
potent opioid peptides from amphibian skin. Br. J. Pharmacol. 1981;
73: 625–631.
38 Chou SH, Wong CH, Chen ST, Wang KT. Enzymatic synthesis of
oligopeptide. Part II. Synthesis of bis(N-protected amino acid)
hydrazides by papain. J. Chin. Chem. Soc. 1979; 26: 11–15.
17 de Castiglione R, Rossi AC. Structure-activity relationships of dermorphin
synthetic analogues. Peptides 1985; 6(Suppl. 3): 117–125.
18 Schiller PW, Nguyen TM-D, Chung NN, Lemieux C. Dermorphin
analogues carrying an increased positive net charge in their ‘message’
domain display extremely high μ-opioid receptor selectivity. J. Med.
Chem. 1989; 32: 698–703.
19 Chaki K, Kawamura S, Kisara K, Sakurada S, Sakurada T, Sasaki Y, Sato T,
Suzuki K. Antinociception and physical dependence produced by [D-
Arg2]dermorphin tetrapeptide analogues and morphine in rats. Br. J.
Pharmacol. 1988; 95: 15–22.
20 Ogawa T, Miyamae T, Murayama K, Okuyama K, Okayama T, Hagiwara M,
Sakurada S, Morikawa T. Synthesis and structure–activity relationships of
an orally available and long-acting analgesic peptide, N(alpha)-amidino-
Tyr-D-Arg-Phe-Me_Ala-OH (ADAMB). J. Med. Chem. 2002; 45: 5081–5089.
21 Lazarus LH, Guglietta A, Wilson WE, Irons BJ, de Castiglione R. Dimeric
dermorphin analogues as μ-receptor probes on rat brain membranes.
J. Biol. Chem. 1989; 264: 354–362.
39 Olma A, Łachwa M, Lipkowski AW. The biological consequences of
replacing hydrophobic amino acid in deltorphin I with amphiphilic α-
hydroxymethylamino acids. J. Pept. Res. 2003; 62: 45–52.
40 Misicka A, Lipkowski AW, Horvath R, Davis P, Kramer TH, Yamamura HI,
Hruby VJ. Topographical requirements for delta opioid ligands:
common structural features of dermenkephalin and deltorphin. Life Sci.
1992; 51: 1025–1032.
41 Fichna J, do-Rego JC, Costentin J, Chung NN, Schiller PW, Kosson P,
Janecka A. Opioid receptor binding and in vivo antinociceptive activity
of position 3 substituted morphiceptin analogs. Biochem. Biophys. Res.
Commun. 2004; 320: 531–536.
42 Jenssen H, Aspmo SI. In Peptide-based Drug Design, Otvos L (ed.).
Humana Press: New York, 2008; 177–186.
43 Frączak O, Lasota A, Kosson P, Leśniak A, Muchowska A,
Lipkowski AW, Olma A. Biphalin analogs containing β3-homo-
amino acids at the 4,4’ positions: synthesis and opioid activity
profiles. Peptides 2015; 66: 13–18.
22 Handa BK, Lane AC, Lord JAH, Morgan BA, Rance MJ, Smith CFC.
Analogues of β-LPH61–64 possessing selective agonist activity at
μ-opiate receptors. Eur. J. Pharmacol. 1981; 70: 531–540.
23 Heck T, Limbach M, Geueke B, Zacharia M, Gardiner J, Kohler HP,
Seebach D. Enzymatic degradation of β- and mixed α,β-oligopeptides.
Chem. Biodivers. 2006; 3: 1325–1348.
24 Johnson LM, Gellman SH. α-Helix mimicry with α/β peptides. Met.
Enzymol. 2013; 523: 407–429.
25 Mollica A, Pinnen F, Costante R, Locatelli M, Stefanucci A, Pieretti S,
Davis P, Lai J, Rankin D, Porreca F, Hruby VJ. Biological active
44 a) Frączak O, Lasota A, Muchowska A, Kosson P, Tymecka D, Misicka A,
Olma A. Dimeric dermorphin analogues containing β3-homo-amino
acids: synthesis, binding affinities and metabolic stability. In
Proceedings of the 24th American Peptide Symposium, Srivastava V,
Yudin A, Lebl M (eds.). American Peptide Society: Orlando, Florida,
USA, 2015; 77–79; b) Frączak O, Lasota A, Muchowska A, Tymecka D,
Misicka A, Olma A. Poster YI-P034: Dimeric dermorphin analogues
containing β3-homo-amino acids: synthesis, binding affinities and
metabolic stability. 24th American Peptide Symposium (20-25 June
2015, Orlando), Book of Abstracts pp. 78.
J. Pept. Sci. 2016; 22: 222–227
wileyonlinelibrary.com/journal/jpepsci