Fine-tuning the π-π Aromatic interactions in peptides: Somatostatin analogues containing mesityl alanine

Article abstract of DOI:10.1002/anie.201106406

Going through the motions: Somatostatin analogues having greater conformational rigidity than somatostatin have been prepared by substituting Phe residues in the native sequence with mesityl alanine (Msa; see structure). The analogues show high affinity f

Full text of DOI:10.1002/anie.201106406

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DOI: 10.1002/anie.201106406
Noncovalent Interactions
Fine-tuning the p–p Aromatic Interactions in Peptides: Somatostatin
Analogues Containing Mesityl Alanine**
Pablo Martꢀn-Gago, Marc Gomez-Caminals, Rosario Ramꢁn, Xavier Verdaguer, Pau Martin-
Malpartida, Eric Aragꢁn, Jimena Fernꢂndez-Carneado, Berta Ponsati, Pilar Lꢁpez-Ruiz,
Maria Alicia Cortes, BegoÇa Colꢂs, Maria J. Macias,* and Antoni Riera*
Understanding noncovalent interactions between aromatic
moieties is essential in medicinal chemistry and lead opti-
mization for drug design. These interactions are fundamental
in controlling diverse phenomena; for example, vertical
stacking interactions provide stability to duplex DNA.[1]
Other important examples include the spike–nucleocapsid
interaction in viruses,[2a] molecular self-assembly in supra-
molecular systems,[2b] and host–guest molecular recognition
events.[2c] Aromatic amino acids strongly contribute to protein
Figure 1. Amino acid sequences of somatostatin and octreotide, and
their respective proposed pharmacophores.
architecture and stability[3,4] as it has been observed in SH3
and WW domains,[5a] and of peptides, including the antimi-
crobial Tachiplesin I[5b] or the pharmacologically important
hormone somatostatin.[6]
The three-dimensional (3D) structure of somatostatin has
been a matter of debate during the last three decades.
Initially, Hirschmann and co-workers[9] postulated the exis-
tence of an interaction between the aromatic residues Phe6
and Phe11. This interaction could enable in the stabilization
of some of the biologically active conformations of the
hormone. A few years later, the same authors detected the
Phe6–Phe11 interaction by NMR spectroscopy,[10a] and
hypothesized that it should be perpendicular (edge-to-face)
rather than parallel (face-to-face).[10a,b] However, van Binst
and co-workers subsequently reported that they were unable
to observe the Phe6–Phe11 interaction by NMR spectroscopy
(either in aqueous solution or in methanol) and that the only
interaction that they could observe was that between Phe6
and Phe7.[11a,b] Attempts to additionally characterize the
somatostatin structure have been unsuccessful. Presently,
there is a consensus that the native structure of somatostatin
in solution is an ensemble of several conformations in
equilibrium, a few of which are partially structured.[12] This
scenario probably explains the failure to obtain detailed
NMR or X-ray data on the 3D structure of the peptide.
To date, most of the work done on somatostatin analogues
has focused on the synthesis of molecules having smaller and
more rigid rings. Some of these compounds have shown
enhanced selectivity and stability.[13] The best examples are
octreotide (Figure 1, right) and lanreotide, the only two
somatostatin analogue drugs on the market.[14] Both of these
compounds have strong affinity for SSTR2 but only moderate
to low affinity for the other receptors. Like most of the
somatostatin analogues currently under research, octreotide
and lanreotide are octapeptides that include part of the
somatostatin pharmacophore[15] and feature a covalent disul-
fide bridge as a surrogate of the proposed noncovalent
interaction between Phe6 and Phe11.
Somatostatin, also known as somatotropin release-inhib-
iting factor (SRIF), is a 14-amino-acid natural peptide whose
sequence is shown in Figure 1 (left). In clinical practice,
somatostatin is currently used as a gastric anti-secretory drug
to treat growth hormone secretion disorders and endocrine
tumors.[7] It is involved in multiple biological functions
mediated by direct interactions between it and at least five
characterized G-protein-coupled receptors, named SSTR1–
5.[8] These receptors differ in their tissue distribution and
pharmacological properties.
[*] P. Martꢀn-Gago, Dr. R. Ramꢁn, Prof. X. Verdaguer,
P. Martin-Malpartida, E. Aragꢁn, Prof. M. J. Macias, Prof. A. Riera
Institute for Research in Biomedicine (IRB Barcelona)
Baldiri Reixac, 10, 08028 Barcelona (Spain)
E-mail: antoni.riera@irbbarcelona.org
maria.macias@irbbarcelona.org
Dr. R. Ramꢁn, Prof. X. Verdaguer, Prof. A. Riera
Departament de Quꢀmica Orgꢂnica, Universitat de Barcelona
Martꢀ i Franquꢃs, 1–11, 08028 Barcelona (Spain)
Prof. M. J. Macias
Instituciꢁ Catalana de Recerca i Estudis AvanÅats (ICREA)
Passeig Lluis Companys 23, Barcelona (Spain)
Dr. M. Gomez-Caminals, Dr. J. Fernꢄndez-Carneado, Dr. B. Ponsati
BCN Peptides, S.A. (Lipotec Group) Barcelona (Spain)
Prof. P. Lꢁpez-Ruiz, M. A. Cortes, Prof. B. Colꢄs
Departamento de Bioquꢀmica y Biologꢀa Molecular, Universidad de
Alcalꢄ de Henares, Facultad de Medicina, Madrid (Spain)
[**] We thank the MICINN (CTQ2008-00763/BQU and BFU2008-
02795), IRB Barcelona, the Generalitat de Catalunya (2009SGR
00901), and BCN Peptides S.A. for financial support. R.R. and P.M.-
G. thank the MICINN for a doctoral fellowship. E.A. and M.J.M.
thank the Consolider RNAREG (CSD2009-00080) for financial
support.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201106406.
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Given recent advances in peptide chemistry which have
greatly facilitated synthesis of large cyclic peptides, we
reasoned that we could introduce point modifications into
the 14-residue scaffold to fine-tune rigidity, specificity, and
stability to produce new analogues that are structurally much
closer to the natural hormone than the octapeptides. In
previous studies,[16] we explored the substitution of Trp8 with
3-(3’-quinolyl) alanine (Qla, both enantiomers), and found
that the corresponding analogues exhibit more conforma-
tional variability than does somatostatin itself. Remarkably,
these analogues were selective for SSTR1 and SSTR3
receptors. Thus, we deduced that structural flexibility is
advantageous for activity in certain receptors and that relative
to the parent compound, these Qla analogues have a greater
proportion of highly flexible conformers in solution.
In seeking new somatostatin analogues that would be
conformationally stabilized (by p–p interactions) relative to
the parent compound, we substituted key amino acids in
somatostatin with nonnatural residues. We prepared various
analogues by replacing the aromatic ring of the phenylalanine
with a mesityl group (2,4,6-trimethylphenyl), that is, by
substituting one Phe with 3-mesityl alanine (Msa, 1; see
Figure 2). We chose Msa based on the higher electronic
density that the methyl groups confer upon the aromatic
moiety, and on the reduced conformational mobility of the
mesityl ring relative to Phe.[17] Hence, we expected that the p–
p interactions between the Msa and the remaining Phe
residues would be stronger than those among the Phe groups
of the parent compound, and envisaged that the intrinsic
rigidity of the Msa amino acid could shift the conformational
equilibrium towards more rigid conformations (relative to
those of the natural compound).
Herein we present how the structural studies confirmed
that the aromatic interactions do exist, and significantly
contribute to both the greater stability and structural rigidity
of our peptide analogues relative to somatostatin. Moreover,
we have also evaluated the interaction of these derivatives
with the five receptors in cellular cultures. We have found that
each of these peptides exhibits a unique profile of strong
affinity and selectivity for one or more of the receptors
SSTR1–5. Furthermore, we have correlated this selectivity to
the presence of aromatic clusters on the basis of the NMR
data, thus paving the way for a rational design of new
efficacious somatostatin-based analogues. We have also
characterized the relative orientation of the aromatic rings
in the clusters, and found that each peptide displays a
particular p–p interaction fingerprint, including parallel,
offset-stacked, and perpendicular orientations as has been
described for proteins.[3]
We obtained Fmoc-l-3-mesityl alanine by following a
procedure previously developed by our group.[19] The four
peptides containing Msa, at either position 6 [l-Msa6,
d-Trp8]-SRIF (2), position 11 [l-Msa11,d-Trp8]-SRIF (3), or
position 7 [l-Msa7,d-Trp8]-SRIF (4), and [l-Msa7]-SRIF (5),
were prepared by solid-phase peptide synthesis on 2-chloro-
trityl chloride resin, using the Fmoc/tBu strategy. Scheme 1
shows the preparation of [l-Msa6,d-Trp8]-SRIF (2). Peptides
3–5 were prepared using the same strategy. When the
nonnatural amino acid was coupled, only 1.5 equivalents
were used.
We initially prepared two different peptides containing
Msa instead of Phe at either position 6 or 11. Additionally, to
increase the physiological stability of the resulting peptides in
blood plasma, we used d-Trp (instead of l-Trp) at position 8
(peptides 2 and 3; Figure 2), a modification known to enhance
stability while maintaining the biological activity of the
peptide.[7a,9,18] Since the aromatic interaction had been
postulated to occur either between residues 6 and 11 (Veber
et al.)[9] or between residues 6 and 7 (Jans et al.),[11a] we were
also interested in the effects of substituting Phe7 with Msa
(peptide 4). The resulting analogue exhibited outstanding
receptor affinity, which prompted us to study the effects of d-
Trp substitution in this compound; thus, we prepared the same
sequence with l-Trp8 (peptide 5).
Scheme 1. a) 1. Fmoc-l-Cys(Trt)-OH (3 equiv), DIEA (3 equiv),
2. MeOH; b) 1. Piperidine 20% DMF, 2. Fmoc-AA-OH (1.5–3 equiv),
DIPCDI (3 equiv), HOBt (3 equiv), DMF (ꢅ12), 3. Piperidine 20%
DMF, 4. Boc-Ala-OH, DIPCI, HOBT, DMF; c) 1. CH2Cl2/TFE/AcOH
(7:2:1), 2. I2, 3. TFA/CH2Cl2/anisole/H2O (12:7:2:1). Boc=tert-butoxy-
carbonyl, DIEA=diisopropylethylamine, DIPCDI=diisopropylcarbo-
diimide, DMF=N,N’-dimethylformamide, Fmoc=N-(9-fluorenylmeth-
oxycarbonyl), HOBT=1-hydroxybenzotriazole, TFA=trifluoroacetic
acid, TFE=2,2,2-trifluoroethanol
With the purified peptides 2–5 in hand, we first measured
the selectivity of each one for each of the five receptors
(SSTR1–5) in binding assays using stable CHO (Chinese
hamster ovary) cell lines. The efficacy of the interaction
Figure 2. The new somatostatin analogues 2–5 having l-3-mesityl
alanine (Msa).
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Table 1: Affinity of somatostatin, [d-Trp8]-SRIF, octreotide, and peptides 2–5 to receptors SSTR1–5. Ki values [nm] ÆSEM.[a]
SSTR1
SSTR2
SSTR3
SSTR4
SSTR5
t1/2 [h]
Somatostatin (SRIF)
[d-Trp8]-SRIF
Octreotide
[l-Msa6,d-Trp8]-SRIF (2)
[l-Msa11,d-Trp8]-SRIF (3)
[l-Msa7,d-Trp8]-SRIF (4)
[l-Msa7]-SRIF (5)
0.43Æ0.08
0.32Æ0.11
300Æ85
0.0016Æ0.0005
0.001Æ0.0007
0.053Æ0.011
4.55Æ0.66
0.53Æ0.21
0.61Æ0.02
15.2Æ5.9
0.78Æ0.1
1.31Æ0.2
7.49Æ0.63
>103
0.74Æ0.07
5.83Æ0.44
>103
0.23Æ0.04
0.46Æ0.24
11.53Æ1.91
0.36Æ0.003
0.73Æ0.19
>103
2.75
19.7
200
26
41
25
3.08Æ0.9
3.35Æ1.32
0.33Æ0.09
4.17Æ1.45
4.70Æ0.92
0.14Æ0.06
>103
0.0024Æ0.001
0.019Æ0.009
>103
28.72Æ6.9
>103
5.2
[a] Numbers in bold represent data in close proximity to (black) or below (grey) the SRIF values.
against each receptor was assessed in competitive assays using
the membranes of the cultured cells and 125I-labeled soma-
tostatin. Somatostatin, [d-Trp8]-SRIF, and octreotide were
used as controls (Table 1).
the obtained structural results were consistent with the NMR
data.
We first analyzed the structural effects of replacing Phe6
with Msa. Analysis of the 2D NOESY spectrum of [l-Msa6,
d-Trp8]-SRIF (2) suggested the presence of a well-defined
structure in solution (Figure 3). The dominant conformer of 2
[l-Msa6,d-Trp8]-SRIF (2) showed high affinity towards
SSTR3 and SSTR5; in fact, its dissociation constants (Ki) for
these receptors are similar to those of somatostatin, which are
20 to 30 times lower than those of octreotide (Table 1).
[l-Msa11,d-Trp8]-SRIF (3) exhibited high affinity for SSTR5,
and significant affinity (although lower than that of somato-
statin) towards SSTR1, SSTR2, and SSTR3 (Table 1).
[l-Msa7,d-Trp8]-SRIF (4) showed striking affinity for
SSTR2—even higher than that of octreotide. Its inhibition
constant for SSTR2 (1.5 times that of natural SRIF) compares
very favorably to that of octreotide (33 times lower than that
of SRIF). Moreover, 4 showed impressive affinity towards
SSTR1, in remarkable contrast to octreotide. Finally
[l-Msa7]-SRIF (5) which lacks d-Trp8, shows a similar profile
than 4 albeit with higher Ki values (Table 1).
To evaluate the structural effects of these site-directed
modifications and subsequently correlate them with the
biological activity observed, we used NMR spectroscopy to
analyze the conformations of these four peptides in aqueous
solution and then compared each one to that of somatostatin.
In all cases proton resonance assignments were identified
using two-dimensional (2D) TOCSY and NOESY homo-
nuclear experiments.[20a] Somatostatin, as deduced from the
pattern of the NOE data, populates several conformations in
solution. All the analogues (2–5) showed more intense NOE
data than did somatostatin, although the majority of the peaks
present in the NMR spectra of these analogues are also
detected in the parent compound. This indicates that each
analogue is structurally similar to one of the characteristic
conformations of the hormone in solution. As shown in the
structures below, the side-chain orientation of peptide 2 is
very different from that of the remaining peptides, thus
explaining why the restraints observed in somatostatin cannot
be fitted to a unique conformation.[12]
Each of the well-defined two-dimensional spectra of
compounds 2–5, enabled us to characterize their main
conformation in solution using the software Crystallography
& NMR System (CNS).[20b] To generate the list of exper-
imental restraints for the calculation, the volume of all
assigned peaks was integrated, and then transformed into
distances. Three sets of calculations (120 structures each)
were run until the best match between assignments and final
structures was obtained. After performing the calculations, all
Figure 3. Superimposition of 28 minimum-energy conformers of
[l-Msa6,d-Trp8]-SRIF (2) as calculated based on NMR data using the
backbone and the side chains of residues 6 to 11 for the fitting.[22]
contains a cluster of three aromatic rings (residues 6, 7, and
11) defined by a large set of NOE interactions among them.
As expected, the two aromatic rings of Msa6 and Phe11 are in
close proximity because of a strong face-to-face p–p inter-
action.[21] Numerous contacts between residues Lys9 and
d-Trp8 are also observed in this peptide.
Peptide [l-Msa11,d-Trp8]-SRIF (3) is also highly struc-
tured. A superimposition of 30 selected low-energy con-
formers of this peptide reveals that the region containing
residues Phe6-Phe7-d-Trp8-Lys9-Thr10-Msa11 is well
ordered (root-mean-square deviation (RMSD) value of
0.384 for the backbone; Figure 4). Peptide 3 also exhibited
a strong p–p interaction between the Phe6 and Msa11 rings,
whereby the Phe7 is located on the opposite face of the
molecule. The orientation is defined by contacts between the
Phe6 ring and the Msa11 ring plus its methyl groups. This
aromatic interaction most likely fixes the backbone confor-
mation (a detailed geometric analysis of the p–p aromatic
interaction is given in the Supporting Information). Interest-
ingly, the p–p interaction occurs at the opposite molecular
face relative to that observed in analogue 2. However, the
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Figure 4. The most stable conformers of [l-Msa11,d-Trp8]-SRIF (3) as
deduced based on NMR data.[22]
Figure 6. Superimposition of the 24 minimum-energy conformers of
[l-Msa7]-SRIF (5) that were calculated based on NMR data.[22] (RMSD
relative orientation of Lys9 and Trp8 side chains is the same in
both compounds.
value of 0.7 for the backbone).
As predicted, substituting Phe with Msa either in position
6 or 11 afforded highly structured peptides, which feature an
internal p–p aromatic interaction between the two aromatic
residues.[21] However, consequences of introducing Msa into
the vicinal position 7 were difficult to predict. To this end, we
investigated structures (in solution) of [l-Msa7,d-Trp8]-SRIF
(4) and [l-Msa7]-SRIF (5). As deduced from 2D NMR data
combined with CNS calculations, peptide 4 showed a well-
ordered family of structures showing a hairpin in the region
encompasing residues 6 to 11 (Figure 5). This conformation is
On the basis of the NMR data that we obtained for each
structure, we deduced that the Msa7 residue is not involved in
forming p–p interactions with either Phe6 or Phe11 since it is
located on the opposite face of the molecule. However the
position occupied by the aromatic ring at position 7 (below
the hairpin, peptides 3, 4, and 5; as shown in Figures 4, 5, and
6, respectively) helps to stabilize the p–p interaction between
the aromatic rings in positions 6 and 11. To the best of our
knowledge, peptides 3–5 are the best structurally defined 14-
residue somatostatin analogues described to date. Peptide 4 is
the least flexible among these exhibiting the most highly
structured conformation because of the combined effects of
reinforced aromatic interaction between Phe6 and Phe11 and
the presence of d-Trp8 in the structure. (RMSD = 0.3)
During the development of short peptide analogues in the
1990s, the structural rigidity of compounds containing the
somatostatin pharmacophore was correlated to their affinity
for SSTR2.[23] The outstanding affinity of peptide 4 for SSTR2
is in good agreement with its high rigidity. We reasoned that
the well-defined conformation that we found for 4 is probably
very close to the native conformation of somatostatin when it
binds to SSTR2. Moreover, we also hypothesized that the
significant activity of peptide 4 against SSTR1 is derived from
the p–p interaction between Msa7 and the Phe195 present in
SSTR1, according to the pharmacophore proposed by Kaup-
mann et al.[24] In peptide 4, Msa7 could interact with Phe195
through a reinforced p–p interaction, a scenario that would
explain the fact that this analogue showed greater affinity for
SSTR1 than the parent compound. Thus, in line with the
suggested induced-fit mechanism for SSTR1,[25] the enhanced
aromatic–aromatic interactions between Msa7 and Phe195
would be essential for the affinity of 4 (which is much more
rigid than peptide 5) to receptor SSTR1.
Figure 5. Superimposition of the 35 minimum-energy conformers of
[l-Msa7,d-Trp8]-SRIF (4) that were calculated based on NMR data.[22]
defined by contacts between the aromatic rings of Phe6 and of
Phe11 and by a set of contacts between the side chains of
d-Trp8 and of Lys9; such contacts restrict the hairpin register.
Interestingly, peptide 4 is structurally very similar to peptide 3
although in the former the aromatic interaction is distinctly
edge-to-face, as defined by contacts between the Phe6
aromatic ring with both Phe11 and the side chain of Lys4.
Peptide 5 with Trp8 in its natural configuration was also
sufficiently structured to have its 3D structure determined by
NMR spectroscopy. The 24 calculated minimum-energy con-
formers that fit the experimental data are shown in Figure 6.
As it seen in the figure, this structure also contains the hairpin,
thus indicating that d-Trp8 additionally stabilizes a confor-
mation that already exists in the natural sequence, and it has
been suggested to be essential for the biological activity of
somatostatin.[18] The Trp8–Lys9 interactions are reflected in
the upfield shifted g protons of Lys which are shielded by the
aromatic indole ring.
Peptide 2 (Msa in position 6) was found to have a
completely different selectivity profile than 4, and it was in
good agreement with its distinct 3D structure: it binds SSTR3
and SSTR5 with affinities of the same order of magnitude as
that of somatostatin.
In summary, we obtained two conformationally rigid
somatostatin analogues with complementary selectivity, and
they mimic two of the different conformations that coexist in
the native hormone.
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Finally, we measured the serum stability of our peptides as
well as that of octreotide, somatostatin, and [d-Trp8]-SRIF,
for the sake of comparison. Although the half-life of peptides
2–4 in serum is not as high as that of octreotide, these three
analogues were on average 10 to 20 times more stable than
somatostatin. Their greater stability probably stems from the
presence of unnatural amino acids and, very likely, from the
stronger interaction between the residues at positions 6 and
11 relative to that in the parent compound. As expected, the
half-life of peptide 5 with l-Trp8 is only twice as high as that
of somatostatin. The surprisingly the long half-life of ana-
logue 3 (Msa in position 11) relative to peptides 2 and 4, may
corroborate the fact that the unnatural aromatic residue in
position 11 shields the residue at position 6, as previously
suggested.[10b]
To date, the most conformationally restricted somatosta-
tin analogues have been developed by deletion of amino
acids, reduction of ring size, or formation of a covalent bridge.
These modifications usually improve the intrinsic pharmaco-
logical properties associated with peptide drugs. However, we
have followed a different approach to obtain analogues with
greater rigidity, thus obtaining four new somatostatin ana-
logues with unique activity and selectivity profiles for SSTR1-
5, by fine-tuning the electronic and steric properties of
specific aromatic residues. We have exploited the noncovalent
interactions between the aromatic residues to modulate the
conformational flexibility and provide major advantages in
receptor selectivity and serum stability. By enhancing aro-
matic–aromatic interactions in somatostatin analogues, we
have obtained four peptides with high receptor selectivity and
with restricted conformations that enabled us to determine
their 3D structures by NMR spectroscopy. Furthermore, we
have elucidated the key aspects of the selectivity against the
five somatostatin receptors by simply introducing an unnatu-
ral Msa amino acid in the original sequence. Our results prove
that the modification of noncovalent interactions is a
promising strategy in drug discovery and opens new possibil-
ities for designing unprecedented peptide analogues of
natural compounds.
Received: September 9, 2011
Published online: December 9, 2011
Keywords: conformation analysis · NMR spectroscopy ·
.
noncovalent interactions · peptides · drug design
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Experimental Section
NMR assignment and structure calculation: NMR data were acquired
at 285 K, using trifluoroacetate as a counter-ion at a pH 4.5 on a
Bruker Avance III 600 MHz spectrometer equipped with a z-pulse
field gradient unit. All spectra were processed with NMRPipe/
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volume of all manually assigned peaks in the NOESY spectrum was
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structures were water refined and ranked based on minimum values
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