β2-Homo-amino acid scan of μ-selective opioid tetrapeptide TAPP

Article abstract of DOI:10.3390/MOLECULES25102461

TAPP (H-Tyr-d-Ala-Phe-Phe-NH₂) is a potent, μ-selective opioid ligand. In order to gain further insights into pharmacophoric features of this tetrapeptide, we have performed a β²-Homo-amino acid (β²hAA) scan of the TAPP sequence. To this aim, 10 novel analogues have been synthesized and evaluated for μ-opioid and δ-opioid receptor affinity as well as for stability in human plasma. The derivatives included compounds in which a (R)- or (S)-β²-Homo-Homologue replaced the amino acids in the TAPP sequence. The derivatives with (R)- or (S)-β²hPhe⁴ turned out to bind μOR with affinities equal to that of the parent. β²hAAs in position 1 and 3 resulted in rather large affinity decreases, but the change differed depending on the stereochemistry. β²-Homologation in the second position gave derivatives with very poor μOR binding. According to molecular modelling, the presented α/β-peptides adopt a variety of binding poses with their common element being an ionic interaction between a protonable amine of the first residue and Asp147. A feature required for high μOR affinity seems the ability to accommodate the ring in the fourth residue in a manner similar to that found for TAPP. Contrary to what might be expected, several compounds were significantly less stable in human plasma than the parent compound.

Full text of DOI:10.3390/MOLECULES25102461

molecules
Article
2
β -Homo-Amino Acid Scan of µ-Selective Opioid
Tetrapeptide TAPP
1
,
2
, Piotr Kosson 3 and Aleksandra Misicka 1,*
Dagmara Tymecka * , Piotr F. J. Lipi n´ ski
1
Faculty of Chemistry, University of Warsaw, Pasteura 1, 02–093 Warsaw, Poland
Department of Neuropeptides, Mossakowski Medical Research Centre Polish Academy of Sciences,
Pawi n´ skiego 5, 02–106 Warsaw, Poland; plipinski@imdik.pan.pl
Toxicology Research Laboratory, Mossakowski Medical Research Centre Polish Academy of Sciences,
Pawi n´ skiego 5, 02–106 Warsaw, Poland; pkosson@imdik.pan.pl
2
3
*
Correspondence: dulok@chem.uw.edu.pl (D.T.); misicka@chem.uw.edu.pl (A.M.)
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editors: Mariana Spetea, Raffaele Capasso and Andrea Trabocchi
Received: 28 April 2020; Accepted: 22 May 2020; Published: 25 May 2020
ꢀ
ꢁꢂꢃꢄꢅꢆ
Abstract: TAPP (H-Tyr-d-Ala-Phe-Phe-NH ) is a potent,
µ-selective opioid ligand. In order to
2
gain further insights into pharmacophoric features of this tetrapeptide, we have performed a
2
2
β
-Homo-amino acid (
β hAA) scan of the TAPP sequence. To this aim, 10 novel analogues have been
synthesized and evaluated for
plasma. The derivatives included compounds in which a (R)- or (S)-
the amino acids in the TAPP sequence. The derivatives with (R)- or (S)-
µ
-opioid and
δ
-opioid receptor affinity as well as for stability in human
2
β
-Homo-Homologue replaced
β hPhe turned out to bind
2
4
2
µOR with affinities equal to that of the parent.
β
hAAs in position 1 and 3 resulted in rather large
2
affinity decreases, but the change differed depending on the stereochemistry.
second position gave derivatives with very poor
the presented -peptides adopt a variety of binding poses with their common element being an
ionic interaction between a protonable amine of the first residue and Asp147. A feature required for
high OR affinity seems the ability to accommodate the ring in the fourth residue in a manner similar
β
-Homologation in the
µOR binding. According to molecular modelling,
α/β
µ
to that found for TAPP. Contrary to what might be expected, several compounds were significantly
less stable in human plasma than the parent compound.
Keywords: β2-amino acids;
synthesis of β -amino acids
β
2-Homo-amino acids;
µ-opioid receptor; opioid peptides; TAPP; racemic
2
1
. Introduction
There is a rich repertoire of structural modifications that a medicinal chemist can use when
exploring structure-activity relationships of peptide active compounds or working on the improvement
of their physicochemical properties. This repertoire includes, e.g., backbone cyclization, introduction
of peptide bond isosteres, incorporation of d-amino acids,
with substituted or constrained side-chains, and other unnatural amino acids [
a prominent place is occupied by -amino acids ( -AAs) [ ]. The amino acids of this class have
two carbon atoms between the carboxylic and amino groups. With both mono-substitutions and
α
,α
-disubstituted amino acids, residues
1]. In the latter group,
β
β
2,3
poly-substitutions being possible in different combinations and stereochemistries (Figure 1), β-AAs
constitute a family of building blocks of enormous structural diversity.
β
-Amino acids can be used to construct full
possible to create mixed -peptides in which one or more
-amino acids. There are three main structural consequences of introducing a
β
-peptides (solely made of
-residues are incorporated instead of some
-residue into a peptide
-AA elongates the backbone by one methylene unit. Furthermore, it adds one
β-AAs), but it is also
α
/β
β
α
β
backbone. First, each
β
3
3
freely rotating sp -sp C-C bond, which increases the backbone flexibility. Lastly, in topographical
Molecules 2020, 25, 2461; doi:10.3390/molecules25102461
www.mdpi.com/journal/molecules

Molecules 2020, 25, 2461
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terms, the position of side-chains of the residues that follow a
β-residue is shifted toward the C-terminus.
All these changes can have critical impact on activity and other properties (e.g., stability) of a peptide
sequence and this is why β-AAs are useful means in medicinal chemistry research.
Figure 1.
(A) Examples of diverse possible types of β-amino acids. (B) Comparison of absolute
2
3
configuration of α-, β -, and β -amino acids.
Peptides are endogenous ligands for all types of opioid receptors (
Among these receptors, the type ( -opioid receptor, OR) constitutes a major molecular target
for the treatment of pain, but the medicinal potential of the remaining ORs has been increasingly
acknowledged [ ]. Over the years, both natural and synthetic peptides as well as peptide-inspired
µ, δ, κ, and nociceptin) [4].
µ
µ
µ
5
organic analogues have continued to be key compounds in the research on the opioid receptors.
In attempts to decipher structure-activity relationships of opioid peptides, or in search for more
3
stable analogues, the researchers also used
β
-AAs. All-
β
analogues (that is with
β
-Homo-AA in every
].
-amino acids as proline
mimics were used for studying conformational requirements in endomorphin-1/-2 analogues [ ].
Other authors replaced proline for -alanine [ ]. Earlier, cyclic -AAs were applied in studies on
11] and dermorphin [12]. Cardillo et al. conducted a systematic -AA [13] and
-isomer or
-Homo aromatic amino acids were introduced alone
position) of deltorphin I, Leu-enkephalin, and dermorphin were reported by Wilczy n´ ska et al. [
6
The same paper presented several -hybrides of deltorphin I. Alicyclic
α
/β
β
7,8
β
9
β
morphiceptin [10
β
,
β
3
-Homo-AA scan of endomorphin-1 [14], exchanging each single AA in the sequence for its β
3
3
2
β
-Homologue. In another work,
β -Homo and β
or in combination in positions 3 or/and 4 of endomorphin sequence [15
,
16]. Mollica et al. performed a
3
3
β
-Homo-AA scan of a dimeric peptide, biphalin [17]. Several derivatives of this peptide with
β
hPhe
3
or
β
-Homo-p-NO -Phe were reported by Fra˛czak et al. [18]. Dimeric derivatives of dermorphin were
2
3
probed with
β
-hAAs as well [19]. Recently, Adamska-Bartłomiejczyk et al. incorporated
β-AAs
into the structure of a cyclic endomorphin derivative, Tyr-c[D-Lys-Phe-Phe-Asp]NH2 [20]. It is also
polysubstituted -AAs that were used in the opioid field. For example, several recent papers dealt
with endomorphin-1/-2 analogues containing one or more β -AAs [21–25].
β
2,3

Molecules 2020, 25, 2461
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An interesting
µOR selective opioid peptide is TAPP (H-Tyr-d-Ala-Phe-Phe-NH2,
1
) [26].
Structurally, it resembles endomorphin-2 (H-Tyr-Pro-Phe-Phe-NH ) in having Phe-Phe in positions
2
3
and 4, but it is also similar to some enkephalin-based derivatives, such as DAMGO
(
H-Tyr-d-Ala-Gly-N-MePhe-Gly-ol), in having d-Ala in the second position. The TAPP sequence was
3
probed by a
complement their work by conducting a
β
-Homo-AA scan by Podwysocka et al. [27]. In the presented research, we wanted to
2
β
-Homo-AA scan. To this aim, we have synthesized, tested
for receptor affinity, and plasma stability as well as subjected to molecular modelling 10 novel TAPP
2
analogues. In these,
β
α
-AAs in each position were replaced by their
β
-Homologues. Additionally,
2
2
h-m-Tyr was used in the first position. As we employed both (R)-isomers and (S)-isomers of
β
h-AAs,
we were able to probe the effect of backbone expansion with the concomitant retention or reversion of
2
side-chain spatial positioning. In this case, it is worth noting that, in
β
-AAs, the very same spatial
-AAs gives usually different
3
arrangement of substituents at the asymmetric carbon as in
α
-AAs or
β
2
absolute configurations (Figure 1B). In addition, a direct
β
-Homo-counterpart (in terms of side-chain
location) of (S)-Phe is (R)-β hPhe (exceptions are β hCys, β hThr, and β hSer).
2
2
2
2
2
. Results and Discussion
2
.1. Chemistry
The planned modifications of the TAPP structure required preparation of
β
2-Homo-amino acids.
2
Since chiral synthesis of
β
-amino acids is labour-intensive (several stages of synthesis), we decided to
2
start with
β
-amino acids in the racemic form. These were prepared in two-step syntheses (Scheme 1),
). This substrate was either alkylated with methyl iodide to
-methyl cyanoacetate (3a, alanine analogue path) or transformed into Z/E isomers of
-cyano-cinnamates (3b ) via Knoevenagel condensation with aromatic aldehydes (aromatic
analogues path). In the second step, the intermediates 3a were subject to simultaneous one-pot
reduction of nitrile groups and double bonds along with Boc-protection. This was possible due to
using CoCl -NaBH combination in anhydrous methanol with di-tert-butyl dicarbonate as a trapping
starting from methyl cyanoacetate (
form methyl
methyl
2
α
α
–d
–
d
2
4
(
to prevent dimerization into secondary amines) and a protective agent [28
,
29]. Thus, fully N-protected
2
and C-protected racemic β -Homo-amino acids (4a–d) were obtained in moderately good yields.
2
Scheme 1. Synthesis of racemic
β
-Homo-amino acids. Reagents and conditions (i) MeI, K CO3(dry),
2
-
N,N dimethylformamide (DMF), (ii) Ar’CHO, piperidine, MeOH, (iii) CoCl2
·
6H O, NaBH , Boc O,
2 4 2
MeOH(dry)
.

Molecules 2020, 25, 2461
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Furthermore, an attempt was made to separate pure enantiomers (Scheme 2). After C-deprotection,
the acids (5a ) were derivatized by coupling with (S)-( )- -methylbenzylamine. In the case of alanine
and phenylalanine analogues, the resulting diastereoisomeric amide pairs (6a ) were separated with
column chromatography and further hydrolysed with 6M HCl. After purification (ion-exchange
chromatography), the absolute configuration of the products (7a ) was determined by measuring
specific rotation and comparing it to the literature values. Regarding diastereoisomeric pairs of amides
–
d
−
α
–
b
–
b
2
2
of
β h-Tyr and β h-m-Tyr (6c–d), it was possible to separate them using preparative high performance
liquid chromatography (HPLC). However, hydrolysis and isolation of the expected products failed.
Scheme 2. Separation of racemic
TBTU, diisopropylethylamine (DIPEA), DMF, (ii) column chromatography: hexane (or CHCl )/AcOEt,
β
2-Homo-amino acids. Reagents and conditions (i) coupling reagent
3
(
iii) 6M HCl reflux, (iv) ion exchange chromatography (Dowex 50W-X12), NH aq.
3
The planned
were formed from respective
α
/
β
-tetrapeptides (
8
–
17) were then synthesized in solution. First, dipeptides
2
α
- or
β
-Homo amino acid derivatives. Then, the dipeptides were
coupled to yield the tetrapeptides, which was followed by ammonolysis to obtain the desired
2
2
tetrapeptide amides. In the case of analogues containing
β hTyr and β h-m-Tyr, diastereoisomeric
mixtures were used for dipeptide syntheses and, therefore, diastereoisomeric mixtures of tetrapeptides
were obtained. It was possible to separate single isomers by preparative HPLC. The absolute
configurations of these derivatives were assigned based on HPLC retention times, per analogiam to
2
isomers of
β
hPhe-d-Ala-Phe-Phe-NH2 (18
–19), on the assumption that the presence of meta-phenol
or para-phenol groups does not influence the elution order of diastereoisomers. The names of the
analogues for which such an assignment procedure was performed will be further on marked with an
2
2
asterisk: [(R)-β hTyr]-TAPP*, [(S)-β hTyr]-TAPP* etc.
2
.2. Opioid Receptor Affinity
The parent compound (TAPP, 1) and the synthesized α/β-peptides (8–17) were tested for binding
affinity to
radioligand displacement assay with selective radioligands. The results are presented in Table 1 (
and Table SM-BIN-1 in the Supplementary Materials ( OR) as half-maximal inhibitory concentration
IC ) with standard errors of the mean (S.E.M).
µ-opioid and
δ-opioid receptors. The determinations were performed by a competitive
µOR)
δ
(
50
The parent peptide (TAPP) was found to have high affinity for
low binding to OR, which is consistent with the data in the original TAPP report by Schiller et al. [30].
Introduction of an additional -CH - unit into the TAPP backbone resulted in diverse changes in OR
µOR (IC50 = 5.1
±
3.5 nM) and only
δ
µ
2
affinity, depending on the position where the backbone was expanded. Position 4 does not seem
2
4
sensitive for this modification at all. Derivative [(R)-
β
hPhe ]-TAPP (12) exhibited IC50 of 1.9 ± 2.4 nM
,
which is equal to (not significantly different than) the value found for TAPP. In position 3, the additional
2
3
methylene ([(R)-
β hPhe ]-TAPP, 11) brought about a more than 10-fold decrease in µOR binding.
An even greater drop in affinity was observed for a derivative with the expansion in position 2. For this
2
2
compound ([(S)-
β hAla ]-TAPP, 10) the IC50 value was greater than 1000 nM, which means that it
is at least a 200-times weaker ligand than the parent. The backbone expansion in the first position

Molecules 2020, 25, 2461
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decreased the affinity about 15-times ([(R)- 2
β
hTyr ]-TAPP*,
1
8). However, in this case, the binding
strength may be partially restored if the expansion is accompanied by meta-positioning of the phenol
2
1
group ([(R)-β h-m-Tyr ]-TAPP*, 9).
Table 1. Binding affinity of the studied compounds.
1
IC50 ± S.E.M. [nM]
Compound
2
No.
1
µOR
TAPP
5.1 ± 3.5
Backbone Expansion without Changing the Spatial Positioning of the Side-Chain
2
1
8
9
[(R)-β hTyr ]-TAPP*
77.6 ± 2.4
48.9 ± 2.3
> 1000
2
1
[(R)-β h-m-Tyr ]-TAPP*
2
2
1
0
1
2
[(S)-β hAla ]-TAPP
2
3
1
1
[(R)-β hPhe ]-TAPP
95.4 ± 2.5
2
4
[(R)-β hPhe ]-TAPP
1.9 ± 2.4
Backbone Expansion with Changing the Spatial Positioning of the Side-Chain
2
1
1
1
1
1
1
3
4
5
6
7
[(S)-β hTyr ]-TAPP *
338.8 ± 5.1
11.2 ± 6.3
954.9 ± 2.9
15.5 ± 2.5
7.8 ± 4.0
2
1
[(S)-β h-m-Tyr ]-TAPP*
2
2
[(R)-β hAla ]-TAPP
2
3
[(S)-β hPhe ]-TAPP
2
4
[(S)-β hPhe ]-TAPP
1
IC50 ± S.E.M. Half-maximal inhibitory concentration
±
standard error of the mean. Mean of three determinations
2
3
in duplicate. Radioligand: 0.5 nM [ H]DAMGO.
If simultaneously with the introduction of a methylene unit into the backbone, side-chain
positioning was inverted, the observed trends in affinity were not parallel to those in the former
2
4
series. Position four is the least sensitive one, and the derivative [(S)-
β
hPhe ]-TAPP (17) exhibited
2
similar
µ
OR binding strength as TAPP. Not much worse was the analogue with (S)-
β
hPhe in position
2
3
(
16), which is in contrast to the former series where (R)-
β
hPhe in this position (16) brought a
2
more pronounced decrease in affinity. Introduction of (R)-
with very low affinity (15, IC50 = 954.9
accompanied with an inversion of the side-chain positioning was associated with a large decline in
β
hAla in position 2 produced a derivative
±
2.9 nM). With respect to position 1, backbone expansion
2
1
µ
OR affinity ([(S)-
β
hTyr ]-TAPP*, 13, IC50 = 338.8
±
5.1 nM). Notably, if additionally the phenol group
was switched to the meta-position, the binding strength was restored and equal to that of the parent
2
1
peptide ([(S)-β h-m-Tyr ]-TAPP*, 14, IC = 11.2 ± 6.3 nM).
50
As to the
14 15) did not exhibit any measurable binding for the
in the fourth position accompanied by inversion of the side chain location ([(S)-
brought about a slight improvement in
δ
OR affinity (Table SM-BIN-1), derivatives with modifications in positions 1 and 2 (
8,
9,
10
,
1
3
,
,
δ opioid receptor. Expansion of the backbone
2
4
β
hPhe ]-TAPP, 17
)
δ
OR affinity when compared to the parent peptide, but the IC50
value reads in the high-middle nanomolar range. The remaining three derivatives (11
δOR binding at a level similar to the parent TAPP.
, 12, 16) exhibited
Comparing these results to the work by Podwysocka et al. [27], a disparity of trends is
3
noticed.
β
-Homo-AAs in position 1 and 3 gave completely inactive derivatives, irrespectively
2
of the stereochemistry, while, in our work,
affinity decreases that varied with stereochemistry and [(S)-
a slightly worse binder than the parent compound. On the contrary,
gave a derivative with nanomolar affinity, and, in our work, analogues with
β
-Homo-AAs in these positions produced smaller
µ
OR
2
3
β
hPhe ]-TAPP can be considered as only
3
β
-Homologation in position 2
2
2
β hAla did not have
3
appreciable
µ
OR binding. Furthermore, in our report, derivatives with
β
h-AA in the fourth position
2
4
are essentially equipotent to the parent, but, for
times was reported.
β -hAA analogues, an affinity decrease by about six

Molecules 2020, 25, 2461
6 of 18
2
.3. Molecular Modelling
In order to understand the observed structure-activity trends in terms of ligand-receptor
interactions, the compounds and 8–17 were docked into the -opioid receptor structure (PDB
accession code: 6DDF [31]) using AutoDock 4.2.6 [32].
1
µ
The best scored pose for the parent TAPP (
1
) is presented in Figure 2A. Docking predicts that TAPP
1
binds
µOR with the N-terminal Tyr directed toward the intracellular part of the receptor. The complex
1
is stabilized by a canonical ionic interaction of protonated Tyr amine with Asp147. The phenol group
of this residue is involved in hydrogen bonding with His297. Furthermore, the aromatic ring forms
several dispersive (
π
-alkyl) contacts with side chains of Met151, Ile296, and Val300. At the C-terminus,
4
the terminal amide interacts with Thr218 via the hydrogen bond. The Phe is located in a hydrophobic
subsite formed by several residues of transmembrane helix 3 (TM3) and extracellular loops 1 and 2
(
ECL1 and ECL2). There the aromatic ring participates in dispersive (
π
-alkyl) interactions with Trp133,
3
Ile144, and Cys217. The Phe is positioned close to Asn127 and His319. Some other receptor residues
present in the vicinity of TAPP are shown in Figure 2B. Furthermore, an intramolecular hydrogen bond
between C=O of d-Ala and N-H of Phe was predicted by docking.
2
4
Figure 2. µOR binding mode of the parent compound (TAPP, 1) as predicted by docking. (A) The
peptide (green sticks) in the binding site of the receptor (yellow). Side chains of only several residues
are shown. (B) The interactions scheme.
This binding mode is highly similar (Figure 3) to the one experimentally found for DAMGO in
DDE and 6DDF structures [31]. In particular, both modes share (i) the canonical ionic interaction with
6
1
4
Asp147, (ii) similar positioning of Tyr ring, and (iii) location of Phe ring in the same hydrophobic
2
subsite. On the other hand, the d-Ala in TAPP is shifted more towards TM7 and to the binding site
4
outlet when compared to DAMGO. The placement of TAPP’s Phe aromatic ring is also similar to the
location of aromatic rings in small molecular ligands like BU72 (as found in crystallography [33]) or
fentanyl (as found by molecular modelling [34,35]).
For the herein reported α/β-peptides (8–17), the docking predicts that they do not necessarily
adopt the binding mode found for TAPP. The obtained binding poses are presented in Figure 4 and
they are summarized in detail in Table SM-MOD-1 in Supplementary Materials. A feature common for
all studied derivatives is the presence of the canonical ionic interaction with Asp147. On the other
1
hand, the side chain of Tyr may be displaced in comparison to TAPP or DAMGO, participating in
different sets of interactions. Furthermore, in some cases, docking predicts that the peptide bond
elements may form hydrogen bonding to Tyr148. The aromatic ring in position three can be located
either closer to TM7 (as in TAPP) or closer to ECL2 and TM2. With regard to position four, for majority
of the derivatives, the aromatic ring is situated in the hydrophobic pocket formed by TM3, ECL1,
and ECL2, as in the case of TAPP and DAMGO. However, the position of the ring can be more or less

Molecules 2020, 25, 2461
7 of 18
displaced in comparison to these ligands and utterly different positions are found as well. Furthermore,
the interactions of the C-terminal amide are also predicted to vary depending on the derivative.
Figure 3. Comparison of the binding modes of DAMGO (from 6DDF PDB structure [31], pink sticks)
and TAPP (from docking, green sticks). The picture is oriented in the same projection as Figure 2.
Only several side-chains of the receptor binding site (yellow sticks) are shown.
Among this diversity of binding poses, it is hard to establish a coherent relationship between the
modification in the peptide structure and the change in the peptide-receptor interactions. The considered
expansions of the backbone affect, in most cases, more than one interaction site. Unfortunately,
in quantitative terms, the scoring function used for docking is not able to provide a predictive tool for
foreseeing the effects of such modest structural changes since no correlation was found between the
scoring value and the experimental affinity (Figure SM-MOD-1 and SM-MOD-2 in Supplementary
Materials). The analysis of the binding poses in qualitative terms allowed however for detection of an
4
interesting relationship between the position of the Xxx side chain (according to docking) and the
affinity. The derivatives with the worst binding results are predicted to have the aromatic ring of the
fourth residue significantly displaced when compared to DAMGO or TAPP. This can be quantitatively
expressed in the form of a correlation equation (Figure 5A) where the independent variable standing
4
for the ring displacement is the root mean square deviation (RMSD) of Xxx atoms’ positions of a
considered derivative when compared to DAMGO in the 6DDF structure [31]. It turns out that this
2
computational value correlates with the experimental affinity with a coefficient of determination (R )
of 0.67, which can be considered a fairly good explanatory power for such a model.
4
We were then curious to see whether this relationship (of Xxx position and affinity) might have
some general validity. In order to check it, we have docked another 12 H-Tyr-Xxx-Yyy-Phe-NH2
derivatives (Xxx = Ala, d-Ala,
β
-Ala, N-MeAla, N-MeGly, Yyy = Phe or Trp) that were experimentally
4
tested for
µ
OR affinity by Perlikowska et al. [
9
]. For these compounds, the Xxx ring RMSD correlated
2
against affinity with R = 0.60 (Figure 5B). In light of these results, it seems reasonable to conclude that,
for closer or more remote TAPP derivatives, a critical requirement for high
place the Xxx ring in the manner similar to that of DAMGO (that is, in the hydrophobic subpocket
formed by residues of TM3, ECL1, and ECL2).
µ
OR affinity is the ability to
4
2
1
A pair of derivatives that deserves a closer structural look are [(S)-
β
hTyr ]-TAPP*
2
1
(13
,
IC50 = 338.8 ± 5.1 nM) and [(S)-
β
h-m-Tyr ]-TAPP* (14, IC50 = 11.2
±
6.3 nM). In this case, the minute
difference in positioning of the phenol group (para- vs. meta-) is associated with over a thirty-fold
difference in affinity. Our modelling seems to provide explanation for this effect. Figure 6 gives
2
a comparison of the binding poses of 13 and 14
.
β
-Homologation in the first position backbone
accompanied with the inversion of the side chain spatial positioning causes the peptide 13 to adopt

Molecules 2020, 25, 2461
8 of 18
the binding pose of an apparently higher energetics than the parent. This can be associated with an
additional rotor being penalized for entropy and no novel interactions being created. On the other
2
1
hand, if the phenol group is shifted to the meta-position of the aromatic ring ([(S)-
β h-m-Tyr ]-TAPP*,
1
4), it allows for forming an H-bond to backbone carbonyl of Ile296 and this restores the affinity.
Figure 4. Binding modes of compounds 8–17 as predicted by docking. The pictures are oriented
in the same projection as Figure 2. Only several side-chains of the receptor binding site (yellow
1
2
1
2
2
sticks) are shown. (
A
) [(R)- 2
β
hTyr ]-TAPP*,
8
, (
B
) [(R)-
β
h-m-Tyr ]-TAPP*,
9
, (
C
) [(S)-
β
hAla ]-TAPP,
) [(S)-
2h-
) [(R)- 2
β
hPhe ]-TAPP, 11, (
3
E
) [(R)-
β
2
hPhe ]-TAPP, 12, (
4
F
) [(S)-
β
2
hTyr ]-TAPP*, 13, (
1
G
β
10, (
D
1
2
2
2
3
2
4
m-Tyr ]-TAPP*, 14, (H) [(R)-β hAla ]-TAPP, 15, (I) [(S)-β hPhe ]-TAPP, 16, (J) [(S)-β hPhe ]-TAPP, 17.

Molecules 2020, 25, 2461
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Figure 5. Correlation between the displacement of the ring of the fourth residue predicted by docking
and the experimental OR affinity. The relationship for ( ) the -peptides reported in this work,
) derivatives reported in Reference [
µ
A
α/β
4
(B
9
]. The ring displacement is expressed as the RMSD of Xxx ring
4
in the considered derivative (as predicted by docking) compared to N-Me-Phe ring in DAMGO (6DDF
structure [31]). The points marked with dotted texture are arbitrarily chosen 5.82 for pIC50 being less
than 6.
2
1
Figure 6. Comparison of binding poses of compounds [(S)-
[
β
hTyr ]-TAPP*, 13 (orange) and
2
1
(S)-β h-m-Tyr ]-TAPP*, 14 (light blue). Only the first residues of the peptides are shown. The receptor
is partially represented as a surface representing the binding site. Only Ile296 side-chain (yellow sticks)
is explicitly shown.
2
.4. Stability Against Proteolysis in Plasma
The parent peptide (1) and the analogues (8–17) were tested as to stability against proteolysis
in human plasma by a HPLC/MS method [19]. The results are graphically presented in Figure 7
and the representative HPLC chromatograms are given in Figures SM-STAB-1 to SM-STAB-22 in
Supplementary Materials. TAPP turned out to be resistant to proteolysis. After 96 h, more than 90% of
the initial peptide concentration (C96h > 90%) remained in the test sample. This is in marked contrast
to endomorphin-2 (Tyr-Pro-Phe-Phe-NH ) that, in the very same conditions, is rapidly degraded with
2
half-life time (T1/2) of only 30 min. For TAPP, the first proteolytic cleavage occurs at the C-terminal
amide (deamidation).
Several of the studied analogues were much less stable when compared to the parent compound.
2
2
In particular, replacement of d-Ala (
1
) for both (S)-
β
hAla (10) or (R)-
β
hAla (15) in position 2 gave
2
compounds prone to proteolysis with T1/2 of about 4.5 h. Derivatives with
β
hPhe in the third
position were also less stable than the parent, but different rates of cleavage were observed for the

Molecules 2020, 25, 2461
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2
3
stereoisomers. [(R)-
case of [(S)-
β hPhe ]-TAPP (11) was found to have half-life time of about 40 h, while, in the
2
3
β hPhe ]-TAPP (16), more than 65% of the initial peptide concentration remained in the
test solution after 96 h. When modifications in the fourth position are considered, again the isomer
2
with (R)-
β
hPhe (12) was less stable (C96h = 55%) than the peptide with reverse configuration in this
position (17, C96h ~ 90%). The latter was equally resistant to proteolysis as the parent. All derivatives
2
1
with
and the second residue (the site of the first proteolytic cleavage).
On the contrary, for the analogues modified in the first position, the first cleavage occurred at the
β -Homo-amino acids in positions 2, 3, and 4 were cleaved at the peptide bond between the Tyr
C-terminal amide (deamidation) in the case of the parent compound. All they (8, 9, 13, 14) exhibited
stability similar to TAPPs.
Figure 7. Results of stability determinations for compounds
1 and 8–17 expressed as percent of the
initial peptide concentration remaining in the test solution at a certain time-point. Each point is an
average result ± standard deviation calculated from three independent experiments.
3
. Materials and Methods
3
.1. Chemistry
All materials (solvents and reagents) were purchased from commercial suppliers and used without
further purification. The NMR spectra were recorded on a Varian Unity Plus 200 spectrometer operating
1
13
at 200 MHz for H-NMR and 50 MHz for C-NMR. The spectra were measured in CDCl or CD OD
3
3
or acetone d and are given as
δ values (in ppm) relative to TMS. Melting points were determined on a
6
Melting Point Meter KSP1D (A. Krüss Optronic, Hamburg, Germany). TLC analyses were performed
on silica gel plates (Merck Kiesegel GF254, Merck, Darmstadt, Germany) and visualized using UV
light or iodine vapour or ninhydrin test. Column chromatography was carried out (at atmospheric
pressure) using Silica Gel 60 (230–400 mesh, Merck, Darmstadt, Germany) using appropriate eluents.
The crude final peptides were purified using reversed-phase high performance liquid chromatography
(
RP-HPLC) on a preparative C-12 column (Phenomenex, Jupiter 4u Proteo 90A, AXIA 250 × 21.20 mm
using 0.1% trifluoroacetic acid (TFA) in water/acetonitrile as a solvent system with UV detection
214 nm). The peptide purity was estimated by analytical HPLC using a C-12 column (Jupiter 4u Proteo
0A, 250 4.6 mm) and the same solvent system and UV detection as above, and then confirmed in a
)
(
9
×
second solvent system 0.1% TFA in water/methanol. High resolution mass spectra (also low resolution)
were acquired on the Shimadzu LCMS-IT TOF mass spectrometer with electrospray ionization (ESI).
2
3
.2. Synthesis of β -Homo-Amino Acids
3
.2.1. Methyl α-methyl-cyanoacetate (3a)
To a stirred solution of methyl cyanoacetate
2 (30 mmol, 1 equiv) and anhydrous potassium
carbonate (30 mmol, 1 equiv) in dimethylformamide, methyl iodide (30 mmol, 1 equiv) was added.

Molecules 2020, 25, 2461
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Then the reaction mixture was stirred overnight. Next, the reaction mixture was diluted with water
and extracted with diethyl ether (4 ). The combined organic layers successively washed with water
), brine solution (3 ), dried over anhydrous Na SO , and evaporated under vacuum. The crude
compound was purified by column chromatography on silica gel (hexane/ethyl acetate). Pure product
×
(2×
×
2
4
3
a was obtained as colourless, low-density oil (1.42 g, 42%).
1
H NMR (200 MHz, CDCl3) δ: 3.82 (s, 3H), 3.42 (q, J = 7.6 Hz, 1H), 1.37 (d, J = 7.6 Hz, 3H).
13
C NMR (50 MHz, CDCl ) δ: 165.9, 117.35, 53.24, 30.57, 15.30.
3
3
.2.2. General Procedure (GP1) of Methyl α-cyano-cinnamates Synthesis via Knoevenagel
Condensation (3b–d)
The benzaldehyde (30 mmol, 1 equiv) or 3- or 4-hydroxybenzaldehyde, methyl cyanoacetate
2
(30 mmol, 1 equiv) and piperidine (0.2 mmol, 0.007 equiv) were heated under reflux for 8–12 h
in methanol. The reaction mixture was allowed to reach room temperature. Then volatiles were
evaporated. The crude compounds 3b were purified by column chromatography (hexane/ethyl
–
d
acetate) or crystallization from methanol.
Methyl α-cyanocinnamate (3b)
The desired product was isolated by column chromatography (hexane/ethyl acetate) or
◦
◦
crystallization (methanol) as white solid (4.98 g, 89%), mp = 90–91 C [lit. 89–90 C] [36].
1
H NMR (200 MHz, CDCl3) δ: 8.27 (s, 1H), 8.06–7.88 (m, 2H), 7.60–7.42 (m, 3H), 3.94 (s, 3H).
C NMR (50 MHz, CDCl ) δ: 162.94, 155.28, 133.38, 131.33, 131.06, 129.25, 115.41, 102.47, 53.36.
13
3
Methyl α-cyano-4-hydroxycinnamate (3c)
The desired product was isolated by crystallization (twice from methanol) as a pale yellow solid
◦
◦
(
4.63 g, 76%), mp = 210–212 C [lit. 208–210 C] [37]
1
H NMR (200 MHz, CD OD) δ: 8.21 (s, 1H), 7.98–7.88 (m, 2H), 6.95–6.87 (m, 2H), 3.88 (s, 3H).
C NMR (50 MHz, CD OD) δ: 165.09, 164.46, 156.13, 135.17, 124.49, 117.30, 115.45, 98.58, 53.50.
3
1
3
3
Methyl α-cyano-3-hydroxycinnamate (3d)
The desired product was isolated by crystallization (twice from methanol) as a yellow solid (4.81 g
,
◦
◦
7
9%), mp = 139–140 C [lit. 142–143 C] [38]
1
H NMR (200 MHz, CD OD)
δ: 8.21 (s, 1H), 7.47–7.34 (m, 3H), 7.05–6.99 (m, 1H), 3.90 (s, 3H).
3
13
C NMR (50 MHz, CD OD)
δ: 164.41, 159.42, 156.61, 134.21, 131.48, 124.04, 121.93, 117.90, 116.48,
3
1
03.57, 53.91.
2
3
.2.3. (R,S)-N-Boc-β -Homo-Alanine Methyl Ester (4a)
To a stirred solution of the methyl
20 mmol, 2 equiv), CoCl2
70 mmol, 7 equiv) were added gradually over 30 min. The reaction mixture was then stirred overnight
α
-methyl-cyanoacetate 3a (10 mmol, 1 equiv), Boc anhydride
◦
(
(
·
6H O (2 mmol, 0.2 equiv) in dry methanol at 0 C, sodium borohydride
2
at room temperature. Then, triethylamine (10 mmol, 1 equiv) was added and stirring was continued for
0 min. Next, the solvent was evaporated and the residue was diluted with ethyl acetate and extracted
3
with saturated aqueous NaHCO (3
×
). The organic layer was washed with brine solution and dried
3
2
over anhydrous Na SO , and then evaporated under vacuum. The product (Boc-
β
hAla-OMe, 4a
)
2
4
was isolated by column chromatography on silica gel (hexane/ethyl acetate) as solidifying, colorless oil
1.30 g, 60%).
1H NMR (200 MHz, CDCl3)
.43 (s, 9H), 1.12 (d, J = 7 Hz, 3H). C NMR (50 MHz, CDCl )
(
δ
: 4.86 (br s, 1H), 3.71 (s, 3H), 3.54–3.12 (dm, 2H), 2.54–2.36 (m, 1H),
13
1
3
δ
+
: 175.07, 155.77, 79.24, 52.15, 43.18,
3
+
9.58, 28.31, 15.91. LR–MS m/z: 140 [M–Boc + Na] , 240 [M + Na] .

Molecules 2020, 25, 2461
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3
.2.4. General Procedure (GP2) of Aromatic
To a stirred solution of the suitable methyl
Boc anhydride (40 mmol, 4 equiv), CoCl2 6H O (4 mmol, 0.4 equiv) in dry methanol at 0 C, and sodium
β
2-Homo-Amino Acid Synthesis, Boc-
β
2hXaa-OMe (4b
–d)
α
-cyano-cinnamate derivative 3b
–d (10 mmol, 1 equiv),
◦
·
2
borohydride (140 mmol, 14 equiv) were added gradually over 30 min. The reaction mixture was then
stirred overnight. Then, triethylamine (20 mmol, 2 equiv) was added and stirring was continued for
3
0 min. Next, the solvent was evaporated and the residue was diluted with ethyl acetate and extracted
with saturated aqueous NaHCO (3 ). The organic layer was washed with brine solution, dried over
anhydrous Na SO , and then evaporated under vacuum. The crude products 4b were isolated by
×
3
–d
2
4
column chromatography on silica gel (hexane or chloroform/ethyl acetate).
2
(
R,S)-N-Boc-β -Homo-phenylalanine methyl ester (4b)
The desired product (Boc-β
2hPhe-OMe, 4b) was purified by column chromatography (hexane/ethyl
acetate) as a solidifying oil (1.61 g, 55%).
1
H NMR (200 MHz, CDCl ) δ: 7.33–7.13 (m, 5H), 4.86 (br s, 1H,), 3.64 (s, 3H), 3.48–3.16 (m, 2H),
3
1
3
3
1
.10–2.74 (m, 3H), 1.42 (s, 9H). C NMR (50 MHz, CDCl3) δ: 174.61, 155.73, 138.22, 128.80, 128.49,
26.56, 79.38, 51.77, 47.35, 41.48, 35.85, 28.30. LR-MS m/z: 316.2 [M + Na] .
+
2
(
R,S)-N-Boc-β -Homo-O-tert-butyloxycarbonyl-tyrosine methyl ester (4c)
The desired product (Boc- 2
β hTyr(Boc)-OMe, 4c) was purified by column chromatography
(
chloroform/ethyl acetate) as a solidifying oil (2.01 g, 49%).
1
H NMR (200 MHz, acetone d6)
δ: 7.32–7.16 (m, 2H), 7.16–6.96 (m, 2H), 6.14 (br s, 1H), 3.57 (s, 3H),
3
.38–3.20 (m, 2H), 3.0–2.76 (m, 3H), 1.52 (s, 9H), 1.40 (s, 9H), 13C NMR (50 MHz, acetone d6)
δ: 174.59,
1
4
52.66, 150.81, 137.62, 130.71, 122.07, 83.50, 79.38, 51.89, 48.81, 42.84, 35.61, 28.64, 27.83. LR-MS m/z:
32.2 [M + Na] .
+
2
(
R,S)-N-Boc-β -Homo-O-tert-butyloxycarbonyl-meta-tyrosine methyl ester (4d)
The desired product (Boc- 2
β h-m-Tyr(Boc)-OMe, 4d) was purified by column chromatography
(
chloroform/ethyl acetate) as a solidifying oil (1.92 g, 47%).
1
H NMR (200 MHz, acetone d6)
δ: 7.38–7.20 (m, 1H), 7.16–6.90 (m, 3H), 6.14 (br s, 1H), 3.57 (s, 3H),
3
.38–3.24 (m, 2H), 3.0–2.78 (m, 3H), 1.52 (s, 9H), 1.40 (s, 9H). 13C NMR (50 MHz, acetone d6)
δ: 176.59,
1
2
56.37, 150.81, 143.25, 142.08, 130.61), 127.65, 121.93, 118.07, 83.63, 79.10, 51.70, 48.81, 42.84, 35.35, 28.31,
7.80. LR-MS m/z: 432.2 [M + Na] , 841.4 [2M + Na] .
+
+
2
3
.3. Separation of Racemic β -Homo-Amino Acids (7a–b)
Boc-protected
stirred solution of 5a
equiv), and DIPEA (10 mmol, 2 equiv) were added. After stirring the mixture at 0 C for 10 min,
the (S)-( )- -methylbenzylamine (5 mmol, 1 equiv) was added. The reaction mixture was then
stirred overnight (allowing to reach to room temperature). Next, the mixture was diluted with 5%
NaHCO and washed several times with ethyl acetate. The combined organic layers successively
β
2-Homo-amino acids 5a
(5 mmol, 1 equiv) in DMF (10 mL), TBTU (5 mmol, 1 equiv), HOBt (5 mmol,
–b (also 5c–d) were obtained according to the GP4. To a
–
b
◦
1
−
α
3
washed with 5% NaHCO (2
×
), 1M KHSO (3
×
), brine solution (3
×
), and dried over anhydrous
) were
separated by column chromatography (supplemented by an additional re-chromatography of partially
3
4
Na SO and evaporated under vacuum. The resulting diastereoisomeric amide pairs (6a
–
b
2
4
separated fractions) using as eluent CHCl /AcOEt for 6a or hexane/AcOEt for 6b. The individual
3
amide diastereoisomers were heated under reflux in 6M HCl for 12 to 18 h. The reaction progress was
monitored by TLC. After the reaction, the mixture was concentrated, and respective (R)- or (S)-7a–b
enantiomers were isolated using cation exchange ion chromatography (Dowex 50W X12). A 0.2 M
ammonia solution was used as an eluent. After evaporation, white solids of individual isomers were
obtained and dried over NaOH.

Molecules 2020, 25, 2461
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2
3
.3.1. (R)-(−)-β -Homo-alanine, (R)-(−)-7a
2
◦
The desired product (R)-(
lit. 185–187 C] [39]; [α]
H NMR (200 MHz, CD OD) δ: 3.26–3.10 (dd, J = 8.6 Hz, J =12.8 Hz, 1H), 3.10–2.96 (dd, J = 4.8 Hz,
J = 12.8 Hz, 1H), 2.96–2.76 (m, 1H), 1.31 (d, J = 7.0 Hz, 3H). C NMR (50 MHz, CD OD) δ: 176.71,
−
)-
β
hAla was obtained as white solid, mp 186–187
C
◦
20
29
[
− 10.7 (c 1.0, 1M HCl) [lit. [α]
− 11.8 (c 1.0, 1M HCl)] [39].
D
D
1
3
13
3
+
+
4
2.68, 38.18, 15.33. LR–MS m/z: 104.1 [M + H] , 207.2 [2M + H] .
2
3
.3.2. (S)-(+)-β -Homo-alanine, (S)-(+)-7a
◦
C
The desired product (S)-(+)-
β
2hAla was obtained as a white solid, mp 187–188
+ 10.2 (c 0.96, 1M HCl) [lit. [α]29 + 11.6 (c 1.0, 1M HCl)] [39].
◦
20
[
lit. 184–188 C] [39]; [α]
D
D
1
H NMR (200 MHz, CD OD) δ: 3.30–3.12 (dd, J = 8.4 Hz, J = 13 Hz, 1H), 3.10–2.92 (dd, J = 5 Hz,
J = 13 Hz, 1H), 2.92–2.80 (m, 1H), 1.27 (d, J = 7.0 Hz, 3H). C NMR (50 MHz, CD OD) δ: 176.76, 42.88,
3
1
3
3
+
+
3
8.98, 15.43. LR-MS m/z: 104.1 [M + H] , 207.2 [2M + H] .
2
3
.3.3. (R)-(+)-β -Homo-phenylalanine, (R)-(+)-7b
◦
C
The desired product (R)-(+)-
β
2hPhe was obtained as a white solid, mp 210–212
+ 13.8 (c 1.083, 1M HCl) [lit. [α]29 + 11.3 (c 1.0, 1M HCl)] [39].
◦
20
[
lit. 225–226 C] [39]; [α]
D
D
1
H NMR (200 MHz, CD OD) δ: 7.35–7.28 (m, 2H), 7.20–6.87 (m, 3H), 3.12–2.90 (m, 3H), 2.80–2.54
3
(
m, 2H). 13C NMR (50 MHz, CD OD)
δ
: 176.61, 138.22, 129.80, 128.49, 126.56, 46.35, 40.48, 36.85.
3
+
+
LR-MS m/z: 180.1 [M + H] , 359.2 [2M + H] .
2
3
.3.4. (S)-(–)-β -Homo-phenylalanine, (S)-(−)-7b
2
◦
C
The desired product (S)-(
lit. 224–225 C] [39]; [α]
H NMR (200 MHz, CD OD) δ: 7.37–7.28 (m, 2H), 7.30–6.95 (m, 3H), 3.14–2.90 (m, 3H), 2.80–2.56
m, 2H). 13C NMR (50 MHz, CD OD)
−
)-
β
hPhe was obtained as a white solid, mp 212–214
◦
20
29
[
− 9.8 (c 0.835, 1M HCl) [lit. [α]
− 11.0 (c 1.0, 1M HCl)] [39].
D
D
1
3
(
δ
: 177.61, 139.20, 128.80, 128.59, 127.56, 45.35, 41.44, 36.67.
3
+
+
LR-MS m/z: 180.1 [M + H] , 359.2 [2M + H] .
3
.4. Synthesis of the Peptides
All planned tetrapeptides (1, 8–17) were obtained according to the following steps of the synthesis:
(I) preparation of Boc-protected N-terminal dipeptide methyl ester by the GP3, (II) preparation of
Boc-protected C-terminal dipeptide methyl ester using the GP3, (III) hydrolysis of the methyl ester of the
product of step (I) according to GP4, (IV) Boc-deprotection of Boc-protected C-terminal dipeptide methyl
ester (step II) using GP5, (V) coupling of Boc-protected N-terminal dipeptide to C-terminal dipeptide
methyl ester according to GP3, (VI) aminolysis of Boc-protected tetrapeptide methyl ester by the GP6.
(
VII) purification by RP-HPLC, confirmation of purity (
≥
96%) by analytical RP-HPLC and confirmation
) 546.2715 (calcd for
of molecular weight by mass spectrometry (3.1. Chemistry). HR-MS: for TAPP (
C H N O , 546.2711), for [(R)-
for [(S)-
(
1
2
1
2
1
β
hTyr ]-TAPP* (
8
) 560.2885, for [(R)-
β
h-m-Tyr ]-TAPP* (
9
) 560.2885,
30
35
5
5
2
2
2
3
2
4
β
hAla ]-TAPP (10) 560.2893, for [(R)-
12) 560.2889, for [(S)-β hTyr ]-TAPP* (13) 560.2874, for [(S)-β h-m-Tyr ]-TAPP* (14) 560.2879,
β
hPhe ]-TAPP (11) 560.2879, for [(R)-
β
hPhe ]-TAPP
2
1
2
1
2
2
2
3
2
4
for [(R)-
β
hAla ]-TAPP (15) 560.2880, for [(S)-
β
hPhe ]-TAPP (16) 560.2879, for [(S)-
β
hPhe ]-TAPP
and 17
(17) 560.2882 (for
8–
17 calcd for C H N O , 560.2867). The analytical data for compounds
1
8–
31
37
5
5
are summarized in Supplementary Materials (Table SM-SYN-1).
3
.4.1. General Procedure (GP3) for Synthesis of Protected Dipeptides and Tetrapeptides
To stirred solution of Boc-amino acid or Boc-dipeptide acid (10 mmol) in DMF (10mL), TBTU
(
10 mmol, 1 equiv), HOBt (10 mmol, 1 equiv), and DIPEA (30 mmol, 3 equiv) were added. After stirring
◦
the mixture at 0 C for 10 min, the amine component or dipeptide methyl ester (10 mmol, 1 equiv) was
added. The reaction mixture was then stirred overnight (allowing to reach the room temperature). Next,

Molecules 2020, 25, 2461
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the mixture was diluted with 5% NaHCO and washed several times with ethyl acetate. The combined
3
organic layers successively washed with 5% NaHCO (2
×
), 1M KHSO (3
×
), brine solution (3
×
),
3
4
and dried over anhydrous Na SO and evaporated under vacuum. The crude peptides were used for
2
4
the next step without further purification.
3
.4.2. General Procedure (GP4) for Methyl ester Removal
To a stirred solution of Boc-protected dipeptide methyl ester (10 mmol, 1 equiv) in MeOH,
◦
LiOH
·
H O (50 mmol, 5 equiv) was added. The reaction mixture was stirred at 0 C for 4–8 h with
2
TLC-monitoring of the reaction progress. Next, the solvent was evaporated under reduced pressure.
The residue was diluted with water and washed with diethyl ether (2 ×), acidified up to pH = 3 with
1
0% citric acid (aq.), and extracted with ethyl acetate (3 ). The organic layer was dried over anhydrous
×
MgSO , and concentrated under vacuum and used for the next step without further purification.
4
3
.4.3. General Procedure (GP5) for Boc-Deprotection
Boc-protected dipeptide methyl ester (10 mmol, 1 equiv) was deprotected by 1N HCl(g) in ethyl
acetate (8–10 mL). The Boc-tetrapeptide amide was deprotected by 1N HCl(g) in acetic acid (8–10 mL)
at room temperature for 2–3 h (TLC-monitoring). Then, the excess of cold ethyl ether was added to the
reaction mixture. Precipitated product was filtered off, washed with diethyl ether, and used for the
next step without further purification.
3
.4.4. General Procedure (GP6) for Aminolysis of Methyl ester
The Boc–protected tetrapeptide methyl ester (20 mmol, 1 equiv) was dissolved in ammonia
solution in methanol (7N, 10 mL) and stirred for 3 to 10 days. The progress of the reaction was
monitored by HPLC. The solution was evaporated under reduced pressure and the crude product was
used for the next step without further purification.
3
.5. Binding Affinity Determinations
The binding affinity of compounds
1
and 8–17 for µOR and δOR was determined in competitive
radioligand binding assays following the previously described method [19
rat brain Homogenate were incubated at 25 C for 60 mins in the presence of radioligands (0.5 nM)
,
40]. Membrane fractions of
◦
3
3
specific for each receptor (
Prof. G za T th [41]) and the increasing concentrations of the tested compounds (10
each concentration in duplicate). For measuring non-specific binding, 10 M naloxone was used as the
competitor. The assay buffer contained 50mM Tris-HCl (pH 7.4), bovine serum albumin (0.1 mg/mL),
bacitracin (30 g/mL), bestatin (30 M), captopril (10 M), and phenylmethylsulfonyl fluoride (0.1 mM)
µOR: [ H]DAMGO and δOR: [ H]DELT II, obtained as a generous gift from
−
11
−5
up to 10 M,
é
ó
µ
µ
µ
µ
in the total reaction volume of 1 mL. After the incubation, the binding reactions were terminated by
rapid filtration with M-24 Cell Harvester (Brandel/USA) through GF/B Whatman glass fibre strips.
The filters were pre-soaked with 0.5% PEI before harvesting in order to minimize non-specific binding.
Filter discs were placed separately in 24-well plates and immersed with the Optiphase Supermix
scintillation cocktail (Perkin Elmer, Waltham, MA, USA). Radioactivity was measured using a MicroBeta
LS Trilux scintillation counter (PerkinElmer, Waltham, MA, USA). The experiments were repeated
three times in duplicate and the results are presented as mean half-maximal inhibitory concentrations
(
IC ) with standard errors of the mean.
50
3
.6. Molecular Modelling
The compounds
DDF [31]) using AutoDock 4.2.6 [32]. The studied derivatives were sketched in Biovia Discovery
1
and 8–17 were docked into activated µOR structure (PDB accession code:
6
Studio Visualizer [42] and minimized with a Dreiding-like force field. Furthermore, they and the
protein structure were processed in AutoDock Tools 4 [32]. Protonation states were set as expected

Molecules 2020, 25, 2461
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at physiological pH. The G-protein was removed from the receptor structure. The ligands were
considered flexible (except for amide bonds of the backbone) and the receptor was set as rigid.
The docking box was centered around the position of DAMGO in the 6DDF structure [31] and its
size was extended to cover the binding pocket and the extracellular part of the binding pocket entry.
Grids were calculated with AutoGrid and the docking was performed using Lamarckian Genetic
Algorithm with pseudo-Solis and Wets local searches (300 runs). A genetic algorithm was set to work
with a population of 3000 individuals with a maximum number of generations being 37,000, mutation
rate of 0.02, crossover rate of 0.8, and one individual surviving to the next generation. The probability of
local search on the individual was set to 0.1. The results of each run were clustered and representative
poses from several top-scored clusters of each ligand were visually inspected. A criterion for selecting
the binding poses was their conformity to known literature data on ligand-µOR interactions, i.e.,
presence of the interaction of protonated amine in position 1 with Asp147. Molecular graphics were
prepared in PyMOL [43].
In order to validate the relationship presented in Figure 5, a further 12 derivatives [9] were docked
to the µOR structure using the very same procedure as described above.
3
.7. Stability Determinations
The determination of plasma stability was performed according to a previously described protocol
with minor modifications [19]. Human plasma for testing was obtained from a healthy donor.
Compounds
of the plasma (100
before adding 100
1
and
8
µ
–
17 (0.7
µ
mol) were dissolved in water to obtain 1 mL of stock solution. Portions
◦
L) were dispensed into Eppendorf tubes and equilibrated at 37
L of the stock solution of each tested peptide. Samples of the test solution were
±
1 C for 5 min,
µ
taken and analyzed at several time points (0, 1, 2, 3, 4, 5, 6, 12, 24, 48, and 96 h of the experiment).
In order to precipitate the plasma proteins, 400
whereafter shaking (1 min) and cooling in 4 C (5 min) followed. Subsequently, the samples were
µ
L ethanol (98%) was added to the samples,
◦
centrifuged for 10 min at 2000 g. The supernatant (20
µL) was immediately analyzed by HPLC/MS
using the LCMS-2010EV Shimadzu apparatus with a Phenomenex Jupiter 4u Proteo 90A, C12
(
25 cm × 2 mm × 4 µm) column. The chromatography was performed in reversed-phase system
(
solvents A: 0.05% FA in water, B: 0.05% FA in acetonitrile) with a non-linear gradient was used (1–31%
B in 20 min followed by an increase to 97% B from 20 to 30 min) at a solvent flow of 0.5 mL/min.
The activity of the plasma was confirmed by using endomorphin-2 (0.7 M/mL) as a control sample.
µ
The results of the HPLC/MS analyses allowed for quantitative determination of the concentration
changes of the tested peptides. Furthermore, it was possible to indicate the location of the first
proteolytic cleavage.
4
. Conclusions
In conclusion, we have presented in this paper the results of
Ten novel analogues were synthesized as well as tested for opioid binding and stability. None of the
β
2hAA scan of the TAPP sequence.
new compounds bound the
had equally good affinity as TAPP. On the contrary,
µ
OR better than the parent, but the compounds modified at position four
2
β
-Homologation in the second position gave
derivatives with very low binding strength. If the third position was modified, the (R)-isomer was
2
weaker than TAPP, but, in the case of the (S)-isomer, the decrease was only minor.
β
hTyr in the first
position caused affinity decreases (more pronounced with the (S)-isomer), but interestingly shifting the
2
phenol group to the meta-position (
β
h-m-Tyr) gave restoration of binding strength. It was particularly
2
1
noticeable with [(S)-
0 times lower than that of [(S)-β hTyr ]-TAPP.
The presented -peptides and their parent, TAPP, were docked into the µOR structure.
β h-m-Tyr ]-TAPP whose IC value was statistically equal to that of TAPP and
50
2
1
3
α
/
β
According to modelling, TAPP binds the receptor by a canonical ionic interaction with Asp147
4
and by anchoring the Phe ring in a hydrophobic subsite formed by residues of TM3, ECL1, and ECL2,
3
while placing the Phe ring closer to TM7. For the novel analogues, docking does not predict a uniform

Molecules 2020, 25, 2461
16 of 18
binding mode, but a rather multitude of poses that vary at several subsites. No straightforward
correlation can be found between the site of backbone expansion and the change in receptor-ligand
interactions. There is also no correlation between the experimental binding affinity and the scoring
value. Yet, it is possible to find a relationship between the experimental binding and the positioning
4
of the Xxx aromatic ring and this relationship can be even expressed quantitatively. Furthermore,
this correlation was validated to hold for some opioid tetrapeptides reported earlier by other authors.
With regard to stability in human plasma, TAPP turned out to be fairly stable (C96h > 90% of the
initial concentration). Surprisingly, the
α
/
β
-analogues were not necessarily so. For example, those with
2
(R/S)-
β
hAla in position 2 turned out to have half-life times of less than 5 h. Modifications in position
2
3
produced derivatives that were less stable than the parent. On the contrary, β hAAs in the first
position gave analogues equally stable to the parent.
The presented results enhance our understanding of structure-activity (stability) relationships of
opioid peptides, and, in particular, of the TAPP sequence. Thus, they will be useful for further design
of peptide analgesics.
Supplementary Materials: The following are available online. SM file is divided into sections that contain data
pertaining to particular analysis subjects. Items in each section are independently numbered. The sections are:
SM-SYN (analytical data for the synthesized peptides, 1 Table), SM-MOD (minor data coming from modelling,
1
Table, 2 Figures), SM-STAB (data from stability determinations, 22 Figures).
Author Contributions: Conceptualization, A.M. and D.T. Data curation, D.T., P.F.J.L., and P.K. Funding acquisition,
A.M. Investigation, D.T., P.F.J.L., and P.K. Methodology, D.T., P.F.J.L., and P.K. Project administration, A.M.
Supervision, A.M. Writing-original draft, D.T. and P.F.J.L. Writing-review & editing, A.M., D.T., P.F.J.L., and P.K.
All authors have read and agreed to the published version of the manuscript.
Funding: The European Union Grant NORMOLIFE (LSHC-CT-2006–037733) supported this work.
´
Acknowledgments: The calculations were performed at Swierk Computing Centre, National Centre for Nuclear
´
Research, Swierk, Poland. Prof. Géza Tóth (Biological Research Centre, Hungarian Academy of Sciences, Szeged,
Hungary) is gratefully acknowledged for providing tritiated radioligands. This work was co-financed by the
EU from the European Regional Development Fund under the Operational Programme Innovative Economy,
2
007–2013, and with the use of CePT infrastructure financed by the same EU program.
Conflicts of Interest: The authors declare no conflict of interest.
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©
2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
CC BY) license (http://creativecommons.org/licenses/by/4.0/).
(