Foldamers of bifunctional diketopiperazines displaying a β-bend ribbon structure

Article abstract of DOI:10.1016/j.tetlet.2010.06.043

The synthesis of two oligomers, of a bifunctional diketopiperazine scaffold DKP-1, formally derived from the cyclization of l-aspartic acid and (S)-2,3-diaminopropionic acid, is reported. A trimeric and a tetrameric structure were prepared by solution-phase peptide synthesis (Boc strategy). Conformational analysis of these derivatives was carried out by a combination of ¹H NMR spectroscopy, CD spectroscopy, and molecular modeling, and revealed the formation of β-bend ribbon involving 10-membered H-bonded rings and a reverse turn of the growing peptide chain.

Full text of DOI:10.1016/j.tetlet.2010.06.043

Tetrahedron Letters 51 (2010) 4278–4280
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Foldamers of bifunctional diketopiperazines displaying a b-bend
ribbon structure
Régis Delatouche a, Marco Durini a, Monica Civera b, Laura Belvisi b, Umberto Piarulli a,
*
a Università degli Studi dell’Insubria, Dipartimento di Scienze Chimiche e Ambientali, Via Valleggio 11, I-22100 Como, Italy
b Università degli Studi di Milano, Dipartimento di Chimica Organica e Industriale and Centro Interdipartimentale C.I.S.I., Via Venezian 21, I-20133 Milano, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of two oligomers, of a bifunctional diketopiperazine scaffold DKP-1, formally derived from
Received 15 April 2010
Revised 4 June 2010
Accepted 8 June 2010
Available online 12 June 2010
the cyclization of L-aspartic acid and (S)-2,3-diaminopropionic acid, is reported. A trimeric and a tetra-
meric structure were prepared by solution-phase peptide synthesis (Boc strategy). Conformational anal-
ysis of these derivatives was carried out by a combination of 1H NMR spectroscopy, CD spectroscopy, and
molecular modeling, and revealed the formation of b-bend ribbon involving 10-membered H-bonded
rings and a reverse turn of the growing peptide chain.
Keywords:
Ó 2010 Elsevier Ltd. All rights reserved.
Secondary structure
Conformational analysis
Peptidomimetics
Helix
b-Amino acids
Circular dichroism
Peptides and peptidomimetics composed of repeating units
have been used to stabilize conformationally ordered structures
(so called foldamers)1 and reproduce the secondary structures of
L-aspartic acid and (S)-2,3-diaminopropionic acid, and bearing a
carboxylic acid and an amino functionality.9 When inserted into
an oligopeptide sequence, the DKP-1-scaffold acts as a reverse turn
proteins and more specifically
a
- and 310-helices. In the latter case
inducer. In addition, DKP-1, while being derived from a-amino
the establishment of a network of hydrogen bonds promotes the
formation of 10-membered rings. A particular type of 310-helix is
the b-bend ribbon, which is characterized by a succession of b-
turns forming a linear peptide with a ribbon-like shape.2 b-Bend
ribbons are found in several natural peptides containing repeating
(Pro-X)n units, such as Zervamicin IIA and other peptaibol antibiot-
ics3 (membrane active peptide antibiotics). Its crystal structure
exhibits a final spiral of three b-bends in a ribbon, containing Pro
(or hydroxyproline)—Aib (or isovaline) sequence motifs.4 Toniolo
and co-workers have investigated the conformational preferences
of several peptides containing an alternation of Pro (or substituted
Pro) residues and helix-forming residues (such as Aib) and have
shown by X-ray diffraction, NMR, and CD studies that these com-
pounds indeed adopt a b-bend ribbon structure both in solution
and in the solid state.5 A different approach was followed by other
authors, using conformationally constrained bifunctional pepti-
domimetics such as 5-carboxy oxazolidin-2-one derivatives6 or
tetrahydrofuran7 and oxetane amino acids.8
acids, can be seen as a conformationally constrained dipeptide
formed by two b-amino acids and in particular a (S) b2- and a (S)
b3-amino acid (following Seebach’s nomenclature).10
Prompted by these observations, we decided to explore the
behavior of oligomers of DKP-1. In this Letter we describe the syn-
thesis and a conformational study of two oligomers: namely the
trimer Boc-(DKP-1)3-NHnBu 2, and the tetramer Boc-(DKP-1)4-
NHnBu 3.
The synthesis of the trimer 2 and tetramer 3 was realized in
solution (Boc strategy) starting from the C terminus. All the cou-
pling reactions were performed using Carpino’s reagent (HATU)
and i-Pr2NEt or collidine in DMF or acetonitrile. The highest yields
COOH
β3-amino acid
H
O
N
N
Ph
O
β2-amino acid
We have recently reported the synthesis of a new bifunctional
diketopiperazine scaffold (DKP-1, Fig. 1), formally derived from
NHBoc
DKP-1
* Corresponding author. Tel.: +39 031 238 6444; fax: +39 031 238 6419.
E-mail address: umberto.piarulli@uninsubria.it (U. Piarulli).
Figure 1. Structure of the bifunctional diketopiperazine scaffold DKP-1, highlight-
ing the conformationally constrained b2–b3 dipeptide sequence.
0040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.06.043

R. Delatouche et al. / Tetrahedron Letters 51 (2010) 4278–4280
4279
in the coupling of the 3rd and 4th DKP-1 residues were obtained
using collidine in CH3CN.
NH3
NH3
A
c
The conformational preferences of these compounds in solution
were investigated by 1H NMR and CD spectroscopy and molecular
modeling. Characteristic differences in the NMR spectral parame-
ters for unstructured peptides and peptides in extended and intra-
molecularly hydrogen-bonded conformations have been reported
in different organic solvents. In particular, concentration and tem-
perature dependence of the amide protons can be indicative of
aggregation phenomena, while NOE contacts can be highly indica-
tive of the formation of secondary structures, such as b-hairpin,11
when inter-strand contacts are visible.
ppm
2.6
2.7
2.8
2.9
3.0
3.1
3.2
3.3
H4
H4
C
A
The dimeric compound Boc-(DKP-1)2-NHnBu was analyzed
first, but it showed some degree of aggregation already at the low-
est 2 mM concentration in CDCl3 and no significant NOE contacts.
These data are in agreement with an equilibrium between a non-
hydrogen-bonded and an intermolecularly H-bonded status.
The trimer Boc-(DKP-1)3-NHnBu 2 (Fig. 2) was studied next. In
this case no concentration dependence of the NH signals was found
up to 5 mM in CDCl3, CD3OH, and DMSO-d6 and, for this reason, all
the subsequent conformational studies were performed at this
concentration. The amide proton chemical shifts in CDCl3 were
rather deshielded (7.77–8.63 ppm and 6.16 for the NH-Boc) indi-
cating possible intramolecular hydrogen bonds.
Analysis of the NOESY spectrum in CD3OH revealed the most
interesting contacts, while the experiment in CDCl3 was not con-
clusive for the absence of relevant long-range contacts and in
DMSO-d6, signal overlap hampered the full characterization and
assignment of long-range contacts. Experiments in D2O could not
be performed due to the insolubility of 2 in water or aqueous sol-
vents. In CD3OH, several long-range interactions could be detected
which revealed the formation of two b-hairpin conformations. In
particular, NH3A (the carbamate proton) showed a medium NOE
contact with the protons HA4 , and NH3C showed a weak NOE contact
with the protons H4C (Fig. 3). These contacts are indicative of the
formation of a pseudo-reverse turn and of a 10-membered ring
hydrogen bond between the NH3A and NH3C and the C@O of the car-
boxy-groups of the diketopiperazine residues A and C, respectively.
A 10 ns stochastic dynamic (SD) simulation12 was performed for
compound 2 applying the NH3A/HA4 and NHC3/H4C distance restraints
derived from NOE contacts. During the simulation, the 10-mem-
3.4
ppm
8.6
8.4
8.2
8.0
7.8
7.6
7.4
7.2
7.0
Figure 3. Section of the NOESY spectrum of 2 (5 mM in CD3OH) showing inter-
strand NOEs for H4 and NH3 protons.
bered ring hydrogen bond13 was formed in 40% (DKP-A) and 7%
(DKP-C) of the sampled structures. In summary, in the case of
the trimeric structure 2, no b-bend ribbon structure could be ob-
served by NMR, although molecular modeling results suggest the
possible formation of two turns for the first and third residues (A
and C in Fig. 2).
The tetrameric structure 3 was then investigated (Fig. 4). Titra-
tion of the amide protons was performed by addition of CD3OD to a
solution of 3 in DMSO-d6, and the rate of exchange was measured.
The endocyclic amide protons exchanged completely in 7 min,
while the exocyclic amide protons disappeared within 48 min from
the addition of CD3OD. The carbamate NH lasted for 169 min be-
fore complete disappearance. Apparently the endocyclic NH2s are
not involved in hydrogen bonding, while the exocyclic amide pro-
H1N
H4
H4
H2
O
O
N
C
N
Ph
O
H3N
H
O
O
H
H
O
N
B
N
Ph
HN
H
O
O
H
H
O
N
A
N
Ph
HN
O
Figure 4. Structure of tetramer 3: (a) on the left, sequence of hairpins and
formation of the b-bend ribbon (the blue arrow indicates the NOE contact typical of
the hairpin conformation); (b) on the right, snapshot taken from SD simulation (non
polar H and benzyl groups have been omitted for clarity).
tBuO
Figure 2. Structure of trimer 2. Blue arrows indicate the NOE contacts typical of the
hairpin conformation.

4280
R. Delatouche et al. / Tetrahedron Letters 51 (2010) 4278–4280
tons NH3 appear to be intramolecularly bound. A study of the
NOESY in CD3OH and DMSO-d6 (D2O and other aqueous solvents
were not considered due to the insolubility of 3 in these solvents)
showed a medium contact between the carbamate proton NH3A and
H4A. Unfortunately, the same NH3/H4 contacts could not be identi-
fied for the DKP-units B–D, due to the overlap of the H4 protons.
In these units, the strong contacts between H4 of one DKP-unit
and the closer NH3 of the subsequent DKP-unit hid the weaker
NH3/H4 contacts of the same unit. For compound 3 SD simulation
was performed applying distance restraints between the NH3/H4
proton of each DKP-units. The 10-membered H-bonded ring was
formed for the 75% and 44% of the simulation by the DKP-A and
DKP-C, respectively, and less than the 20% by the DKP-B and
DKP-D. According to experimental data and SD analysis, compound
3 is likely to adopt a b-bend ribbon conformation with 10-mem-
bered H-bonded ring on each DKP-unit (Fig. 4).
The ability of oligopeptides 2 and 3 to adopt an ordered second-
ary structure in solution was also evaluated by CD spectroscopy
(Fig. 5). The spectra were measured in methanol (0.2 mM) and
showed a rather different behavior for the two compounds. Oligo-
peptides containing the DKP-1 scaffold which formed 10-mem-
bered H-bonded hairpins displayed typical Cotton effects in their
circular dichroism (CD) spectra.9a In particular, two negative min-
ima, a principal one at 200–205 nm and a second one, less intense,
at about 220 nm, and a negative maximum at 209–215 nm were
found. This pattern is also commonly found in 310-helices and in
particular in b-bend ribbons.
and are indicative of an organized secondary structure involving
repeated turn-inducing units.14
In summary, we have described the synthesis of oligomeric
bifunctional diketopiperazines DKP-1 bearing a carboxylic acid
and an amino functionality and formally derived from the cycliza-
tion of L-aspartic acid and (S)-2,3-diaminopropionic acid. A confor-
mational study of these new foldamers showed that these
diketopiperazines are well pre-organized to induce a reverse turn
of the growing peptide chain with the formation of a 10-membered
hydrogen-bonded cycle and that oligomers of DKP-1 adopt a b-
bend ribbon conformation starting from four units of DKP-1.
Acknowledgments
We thank the European Commission (Marie Curie Early Stage
Research Training Fellowship ‘Foldamers’ MEST-CT-2004-515968)
for financial support and for Ph.D. Fellowships to R.D. We also
gratefully acknowledge Ministero dell’Università e della Ricerca
(PRIN prot. 2008J4YNJY) for financial support and CILEA for com-
puting facilities. We also like to thank Dr. D. Potenza (University
of Milano) for helpful discussions.
Supplementary data
Supplementary data (general experimental methods, typical
reaction conditions and spectroscopic characterization of repre-
sentative compounds, 1H and 13C NMR spectra and conformational
studies for compounds 2 and 3) associated with this article can be
found, in the online version, at doi:10.1016/j.tetlet.2010.06.043.
In the case of the trimeric compound 2, the CD spectrum
showed a rather weak minimum at 198 nm and two strong minima
at 208 and 220 nm. On the contrary, the tetramer 3 showed a
rather strong minimum at 200 nm and a weaker one at 225 nm
with a negative maximum at 215 nm.
References and notes
1. Hill, D. J.; Mio, M. J.; Prince, R. B.; Hughes, T. S.; Moore, J. S. Chem. Rev. 2001, 101,
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This is very similar to the CD spectrum of oligopeptides contain-
ing a central DKP-1 unit, which adopted a 10-membered H-bonded
hairpin conformation,9a and also to other peptidomimetic struc-
tures showing a b-bend ribbon conformation.3b,6b,14 In addition,
the molar ellipticities of the Cotton effects displayed by the tetra-
mer 3 (>130,000 deg cm2 molÀ1 for the peak at 200 nm) are much
more intense than the analogous peaks shown by the hexapeptide-
mimic structure containing DKP-1 and four regular amino acids
Boc-ValAlaDKP-1-ValAla-NHnBu (about 8,400 deg cm2 molÀ1 for
the peak at 201 nm at a 0.5 mM concentration in methanol)9a
2. Crisma, M.; Formaggio, F.; Moretto, A.; Toniolo, C. Biopolymers 2006, 84, 3–12.
3. (a) Duclohier, H. Chem. Biodivers. 2007, 4, 1023–1026; (b) Segalas, I.; Prigent, Y.;
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10. The superscripted number after b specifies the position of the side chain on the
corresponding b-amino acid, see: Hintermann, T.; Seebach, D. Synlett 1997,
437–438.
Tetramer 3
Trimer 2
Boc-ValAlaDKP-1-ValAla-NHnBu
20000
0
x10
-20000
-40000
-60000
-80000
-100000
-120000
-140000
-160000
-180000
-200000
11. For
a review on peptide secondary structures, including b-hairpins see:
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12. MacroModel, version 9.5, Schrödinger, LLC, New York, NY, 2005.
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1995, 35, 103–111.
195
205
215
225
235
245
Wavelength (nm)
Figure 5. CD spectrum of tetramer 3, trimer 2, and the hairpin peptidomimetic Boc-
ValAlaDKP-1-ValAla-NHnBu (Ref. 9a). The data of the latter compound have been
multiplied by a factor 10 to magnify the appearance of the curve.