TAPP analogs containing β3-homo-amino acids: Synthesis and receptor binding

Article abstract of DOI:10.1002/psc.2433

β-Amino acids containing α,β-hybrid peptides show great potential as peptidomimetics. In this paper, we describe the synthesis and affinity to μ-opioid and δ-opioid receptors of α,β-hybrids, analogs of the tetrapeptide Tyr- d-Ala-Phe-Phe-NH₂ (TAPP). Each amino acid was replaced with an l- or d-β³-h-amino acid. All α,β-hybrids of TAPP analogs were synthesized in solution and tested for affinity to μ-opioid and δ-opioid receptors. The analog Tyr-β³h- d-Ala-Phe-PheNH₂ was found to be as active as the native tetrapeptide.

Full text of DOI:10.1002/psc.2433

Research Article
Received: 26 April 2012
Revised: 12 June 2012
Accepted: 19 June 2012
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/psc.2433
TAPP analogs containing b3-homo-amino acids:
synthesis and receptor binding
D. Podwysocka,a† P. Kosson,b A. W. Lipkowskib and A. Olmaa*
b-Amino acids containing a,b-hybrid peptides show great potential as peptidomimetics. In this paper, we describe the synthesis
and affinity to m-opioid and d-opioid receptors of a,b-hybrids, analogs of the tetrapeptide Tyr-D-Ala-Phe-Phe-NH2 (TAPP). Each
amino acid was replaced with an L- or D-b3-h-amino acid. All a,b-hybrids of TAPP analogs were synthesized in solution and tested
for affinity to m-opioid and d-opioid receptors. The analog Tyr-b3h-D-Ala-Phe-PheNH2 was found to be as active as the native
tetrapeptide. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.
Keywords: b3-homo-amino acid; tetrapeptide TAPP analogs; m-opioid agonist peptides; radioligand binding; a,b-hybrids of opioid
peptides
of analogs, only [b3-h-Phe]4TAPP displayed significant affinity to the
m-receptor. All analogs were almost fully resistant to a-chymotrypsin.
Introduction
Over the past three decades, many new bioactive opioid peptides
have been discovered. Opioid peptides are widely known for their
analgesic and psychoactive properties [1–3]. These compounds can
be used as effective medical drugs in anticancer therapy [4,5],
in Alzheimer disease treatment [6] and as painkillers [7,8]. Their
limitations are related to their high sensitivity to endogenous
enzymes, short period of action and low selectivity. These peptides
interact with different subtypes of opioid receptors, such as m, d and
k. Among the three main opioid receptors, m-opioid receptors
(MORs) are perhaps of the greatest clinical importance. They are
involved in pain signal transmission and perception, and
therefore, compounds showing selectivity to MORs can be used
in clinical practice as analgesic drugs. The dissociation of analge-
sia from tolerance using MOR selective agonists is nearly impos-
sible. Indeed, investigations using MOR knockout mice have
demonstrated that both the antinociception and tolerance
effects are MOR-mediated [9].
In the present paper, we examined the receptor binding in
TAPP analogs containing L- and D-b3-homo-amino acids (Figure 2)
in each position (I–IX, Table 2).
We also synthesized two tripeptides containing b3-homo-
amino acid residues in position 1 or 1 and 2, without the D-Ala
residue, to check the influence of the distance between aromatic
rings on bioactivity (X–XI).
Material and Methods
Synthesis of b3-Homo-Amino Acids
Optically pure Boc-b3-homo-amino acids 3 were prepared using
two-step Arndt–Eistert homologation described in the literature.
This methodology is useful for multifunctional amino acids
(e.g. Tyr). The synthesis of Boc-a-diazo ketone intermediates
was easily performed by applying the mixed anhydride methods
[16]. Wolff’s rearrangement of Boc-a-diazo ketones 2 was
initiated by silver benzoate in the presence of silica gel in ethyl
acetate [17] (Scheme 1).
In search for more potent m-selective ligands, the tetrapeptide
Tyr-D-Ala-Phe-Phe-NH2 (TAPP; Figure 1) with high affinity to the
m-opioid receptor has been synthesized [10].
In spite of a D-amino acid residue and amide in the C-terminus
of the peptide, it is not resistant to proteolytic enzymes [11,12].
In 1997 Zadina’s group [13] isolated two tetrapeptides from
bovine frontal cortex: endomorphin-1 (Tyr-Pro-Trp-Phe-NH2)
and endomorphin-2 (Tyr-Pro-Phe-Phe-NH2). Endomorphins show
high affinity and the highest selectivity to the m-opioid receptor
among the peptides found in the mammalian nervous system.
In recent years, much attention has been focused on various
b-amino acids and b-peptides. Investigations include the synthesis
of a,b-hybrids and b-peptides [14]. The substitution of a-amino
acids for their b-isomers in biologically active peptides may result
in increased enzymatic stability and also in a strong influence on
peptide conformation.
Enantiomerically pure N-Boc-protected b3-homo-amino acids
were obtained with high yield (69–95% in two steps; Table 1)
without racemization.
*
Correspondence to: Aleksandra Olma, Faculty of Chemistry, Institute of Organic
Chemistry, Lodz University of Technology, Żeromskiego 116, 90-924 Lodz, Poland.
E-mail: aleksandra.olma@p.lodz.pl
†
Current address at Polpharma SA, ul. Pelplińska 19, 83-200 Starogard Gdański,
Poland.
Previously, we described the conformational and metabolic
consequences of the substitution of Phe with b3-homo-Phe in
position 3 or 4 and N-Me-b3-h-Phe or b3-h-Tic in position 3 of
TAPP [15]. A radioreceptor binding assay showed that in this series
a Institute of Organic Chemistry, Lodz University of Technology, Lodz, Poland
b Mossakowski Medical Research Centre, Polish Academy of Science, Warsaw,
Poland
J. Pept. Sci. 2012
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PODWYSOCKA ET AL.
esters of N-protected tetrapeptides were converted to amides by
ammonolysis (liquid ammonia in methanol). The removal of the
Bzl-protecting group was carried out by catalytic hydrogenation
in acetic acid/methanol using 10% Pd/C as catalyst. The reactions
were controlled using TLC or HPLC.
O
O
H
H2N
N
NH2
N
N
H
H
O
O
HO
Figure 1. TAPP (Tyr-D-Ala-Phe-Phe-NH2).
Peptide Purification and Characterization
Crude peptides were purified by preparative HPLC on a Vydac
218TP C18 column (25 Â 2.2cm; Grace Davison Discovery Sciences,
Deerfield, IL, USA). The binary elution system was (A) 0.05% (v/v) tri-
fluoroacetic acid in water and (B) 0.038% trifluoroacetic acid in ace-
tonitrile/water 90: 10. The linear gradient was 20–50% B over
25 min with a flow of 12 ml/min. UV detection was conducted at
220 nm. Each peptide was >97% pure as determined by analytical
reverse-phase HPLC on a Vydac column (25Â 0.46 cm) using a
Peptide Synthesis
The a,b-hybrids of tetrapeptides (I–XI) were synthesized in solution,
stepwise, starting from the C-terminal dipeptides, using TBTU
(1.1 equivalent) as a coupling reagent in the presence of EDIA
(2.2equivalent). The deprotection of Boc-amino-protecting groups
from dipeptides was accomplished with 2.5N HCl/AcOEt while
from tripeptides and tetrapeptides with 90% TFA/H2O. Methyl
Figure 2. Structures of b3-homo-amino acid.
Scheme 1. Homologation of Boc-amino acids.
Table 1. Protected b3-homo-amino acids
No.
Amino acid
[a]D (CDCl3)
Melting point [ꢀC]
3a
3b
3c
3d
3e
3f
Boc-b3-homo-Ala
À16.2 (c 1) (À14.0 (c 1) [18])
+16.5 (c 1) (+ 16.0 (c 1) [18])
À17.8 (c 1) (À17.5;c1, CH2Cl2 [16])
+17.6 (c 1)
95–98 (White solid [18])
White solid (white solid [18])
White solid (96 [19])
White solid
Boc-b3-homo-D-Ala
Boc-b3-homo-Phe
Boc-b3-homo-D-Phe
Boc-b3-homo-Tyr(OBoc)
Boc-b3-homo-D-Tyr(OBoc)
À6.5 (c1)
117–119
+6.6 (c1)
116–119
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TAPP ANALOGS CONTAINING b3-HOMO-AMINO ACIDS
linear gradient of 30–50% B for 25 min at a flow rate of 1 ml/min,
with UV detection at 220nm. The mass spectra were recorded with
the following spectrometers: a Finningan MAT (Finningan MAT
GmbH, Bremen, Germany) for FAB analysis and a Voyager-Elite (Per-
Septive Biosystem Inc., Framingham, MA, USA) for MALDI analysis.
using two-step Arndt–Eistert homologation. After deprotection
and HPLC purification, the purity of a/b-peptides was higher than
97% (analytical HPLC). The molecular weight of obtained analogs
was confirmed by FAB MS or MALDI MS. The physicochemical
properties of the new compound are presented in Table 2.
Affinities of a,b-peptides, analogs of TAPP, for m-receptors and
d-receptors were determined by the radioreceptor binding
assay method described previously using the radioligands
[3H]-naltrexone and [3H]-deltorphin II as m-receptor-specific
and d-receptor-specific ligands, respectively.
Radioreceptor Binding
New a/b-peptides, analogs of tetrapeptides TAPP, were tested for
m-opioid and d-opioid receptor affinity. The receptor binding assay
was performed using the radioligands [3H]-naltrexone and [3H]-
deltorphin II. Radioreceptor binding assays were performed by the
method described previously [20] and were conducted as follows.
Table 3 shows the binding affinity of a,b-peptide analogs to
d-opioid and m-opioid receptors in comparison with Tyr-D-Ala-
Phe-PheNH2.
The binding affinity results for the synthetic analogs revealed
the following properties: (i) a,b-Hybrids I, II and IV–XI exhibit
the absence of affinity to d-receptors. Only analog III has some,
Radioligand displacement assays
A rat brain membrane preparation containing 0.2–0.5 mg of
protein/ml was incubated for 60 min at 25.5 ꢀC in 50 mM Tris–HCl
buffer (pH 7.4) containing 0.1mg/ml BSA, 30mg/ml bacitracin,
30 mM bestatin, 10 mM captopril and 0.1 mM PMSF in the presence
of 0.5nM [3H]-naltrexone or [3H]DELT II, as well as increasing
concentrations of the analyzed compound (10À11–10À6) in a total
volume of 1 ml. Non-specific binding was determined by the
addition of 10 mM naltrexone. All experiments were performed in
triplicate. Incubation was terminated by rapid filtration with 6 mL
of ice-cold 0.9% NaCl through glass microfiber GF/B Whatman
filters (Whatman, Piscataway, NJ, USA) soaked up in 0.5% PEI using
an M-24 cell harvester (Brandel, Gaithersburg, MD, USA). Filter disks
were then placed on 24-well clear PET 24-well flexible microplates
(PerkinElmer, Waltham, MA, USA) and submerged in 1 ml of
OptiPhase Supermix Cocktail (PerkinElmer). Radioactivity was
counted with a MicroBeta LS Trilux (PerkinElmer) scintillation
counter. Binding curves were fitted using nonlinear regression.
Compound potency was expressed as log IC50 values.
Table 3. Receptor binding of a/b-peptides
Peptide
IC50 [nM]
ma
db
Tyr-D-Ala-Phe-PheNH2 (TAPP)
b3-h-Tyr-D-Ala-Phe-PheNH2
b3-h-D-Tyr-D-Ala-Phe-PheNH2
Tyr-b3-h-D-Ala-Phe-PheNH2
15.5 Æ 6.33 616–797 Æ 7.15
I
Not active
Not active
9.5 Æ 2.9
524 Æ 2.95
>1000
Not active
Not active
794 Æ 5.53
Not active
Not active
Not active
Not active
Not active
Not active
Not active
Not active
II
III
IV Tyr-b3-h-Ala-Phe-PheNH2
V
Tyr-D-Ala-b3-h-Phe-PheNH2
c
VI Tyr-D-Ala-b3-h-D-Phe-PheNH2
>1000
VII Tyr-D-Ala-Phe-b3-h-PheNH2
91.2 (Æ5.54)
95.4 Æ3.72
>1000
c
VIII Tyr-D-Ala-Phe-b3-h-D-PheNH2
IX
X
b3-h-Tyr-D-Ala-b3-h-Phe-PheNH2
b3-h-Tyr- Phe-PheNH2 (X)
602.5 Æ 3.52
>1000
XI
b3-h-Tyr-b3-h-Phe-PheNH2 (XI)
aVersus [3H]-naltrexone.
bVersus [3H]-DT II.
c[13].
Results and Discussion
The new analogs of the opioid tetrapeptide TAPP were obtained
in solution. N-Protected b3-homo-amino acids were synthesized
Table 2. Physicochemical properties of a/b-peptides, analogs of TAPP
No
Peptide
HPLCc
MSa
tR [min]
Purity [%]
Mr
[M + H]+
I
b3-h-Tyr-D-Ala-Phe-PheNH2
b3-h-D-Tyr-D-Ala-Phe-PheNH2
Tyr-b3-h-D-Ala-Phe-PheNH2
Tyr-b3-h-Ala-Phe-PheNH2
13.07
11.82
16.33
7.93
97
98
559. 67
559.67
559. 67
559. 67
559. 67
559. 67
559. 67
559. 67
488.89
488. 6
560.4a
560a
II
III
IV
V
98
560.3a
560.3a
560a
98
e
Tyr-D-Ala-b3-h-Phe-PheNH2
10.39
13.18
10.39
12.35
13.04
8.34
99
VI
VII
VIII
IX
X
Tyr-D-Ala-b3-h-D-Phe-PheNH2
100
97
560a
Tyr-D-Ala-Phe-b3-h-PheNH2
560a
e
Tyr-D-Ala-Phe-b3-h-D-PheNH2
b3-h-Tyr-D-Ala-b3-h-Phe-PheNH2
b3-h-Tyr- Phe-PheNH2
98
560a
99
489/596.3[M + Na]b
98
489a
503,4a
XI
b3-h-Tyr-h-b3-h-Phe-PheNH2
14.23
100
502.6
aFAB MS.
bMALDI MS.
cLinear gradient 30–50% B, 25 min flow rate 1 ml/min.
dLinear gradient 20–80% B.
e[15].
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PODWYSOCKA ET AL.
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albeit weak, d-affinity; similar to TAPP. (ii) In all analogs, the substi-
tution of Tyr-1 with L- or D-b3-homo-Tyr reduces their activity (X) or
gives inactive peptides (I, II, IX and XI). Surprisingly, the tripeptide
without D-Ala (X) binds to m-receptors only 40 times less than native
TAPP whereas the tripeptide containing two b3-homo-amino acids
in positions 1 and 2 (XI) is not active. (iii) The replacement of D-Ala
with b-homo-D-Ala (III) results in the most interesting analog.
This modification gives an analog with m-binding affinity (IC50
;
9.5 Æ 2.9) comparable with TAPP (IC50; 15.5 Æ 6.33). Also the
analog containing b3-homo-Ala in position 2 shows affinity (33
times lower as compared with TAPP) to the m-receptor (IV). The
configuration of b3-homo-alanine in position 2 has a strong
impact on binding affinity. (iv) The incorporation of b3-homo-
Phe in position 3 (V, VI) causes a loss of affinity to m-receptors,
whereas the presence of L- or D-b3-homo-Phe in the C-terminus
position (VII, VIII) decreases affinity to m-receptors only sixfold.
The chirality of b3-homo-Phe in position 3 or 4 has no influence
on binding affinity. It is worth to mention that homologation
of Phe4 residue is detrimental to m-affinity in endomorphin-1
analogs [21].
The present results suggest that an increased distance between
two aromatic rings, or increased flexibility (an additional CH2
group in the main chain of the peptide), can facilitate fitting to
the m-opioid receptor. The analog Tyr-b3h-D-Ala-Phe-PheNH2
may adopt a conformation well tolerated by the m-opioid recep-
tor during the peptide ligand–receptor interaction. This result
can be compared with the one previously achieved with the
synthesis of the endomorphin-1 and 2 analogs containing alicyclic
b-amino acids [22] and piperidine-2-, 3- or 4-carboxylic acids [23] in
position 2. It confirms that homologation of residue 2 in m-selective
tetrapeptides is generally well tolerated and does not cause
significant changes in activity and selectivity.
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Acknowledgement
This work was supported partially by the Lodz University of Tech-
nology grant DS I-18/501/15.
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