Participation of aromatic side chains in diketopiperazine ensembles

Article abstract of DOI:10.1016/j.tetlet.2008.04.156

This study probes the beneficial role of aromatic side chains in peptide self-assembly by choosing four diketopiperazine model systems variably composed of glycine, proline, phenylalanine, and tryptophan residues.

Full text of DOI:10.1016/j.tetlet.2008.04.156

Tetrahedron Letters 49 (2008) 4231–4234
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Tetrahedron Letters
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Participation of aromatic side chains in diketopiperazine ensembles
K. B. Joshi, Sandeep Verma *
Department of Chemistry, Indian Institute of Technology-Kanpur, Kanpur 208 016 (UP), India
a r t i c l e i n f o
a b s t r a c t
Article history:
This study probes the beneficial role of aromatic side chains in peptide self-assembly by choosing four
diketopiperazine model systems variably composed of glycine, proline, phenylalanine, and tryptophan
residues.
Received 29 February 2008
Revised 24 April 2008
Accepted 30 April 2008
Available online 2 May 2008
Ó 2008 Elsevier Ltd. All rights reserved.
The self-assembly process is governed by synergistic participa-
tion of multiple non-covalent interactions. Of the various possibil-
O
O
1
H
N
O
NH
ities, hydrophobic aromatic interactions are known to be the
crucial determinants of stability of many bioinspired self-assem-
bled systems. The significance of these interactions is reflected in
nucleic acid structure stabilization, ligand-receptor interaction,
HN
H
NH
O
1
2
NH
2
and protein structure stabilization. The aromatic interactions rep-
H
N
H
resent a combination of non-covalent contacts which include elec-
O
H
N
H
NH
3
O
trostatic, hydrophobic, and van der Waals’ interactions.
H
In this connection, formation of peptide and protein fibers and
aggregates by invoking favorable hydrophobic interactions is an
H
N
H
O
N
H
O
HN
4
area of contemporary interest. Within the context of protein/pep-
3
tide nanotechnology, aromatic side chains support self-assembled
structures from short peptides5 and remarkably, even aromatic
dipeptides afford ordered nanostructures where the aromatic
interactions are implicated in providing the order and directional-
ity needed for the self-assembly process.6
4
Figure 1. Molecular structures of: (1) cyclo-(Gly-Gly); (2) cyclo-(Trp-Pro); (3) cyclo-
Trp-Trp), and (4) cyclo-(Phe-Phe).
(
,7
works and closely packed fibers leading to filamentous aggregates
Diketopiperazines (DKPs) are interesting model systems for
studying the self-assembly process as they afford a variety of struc-
tures including entangled and elongated aggregates such as rods,
ribbons, helices, and tubules.8 It is possible that these structures
may be modulated by varying the substituents on the DKP ring.
This study involves the synthesis of four synthetic DKPs: cyclo-
(
Fig. 2e and f).
Such dichotomy in morphologies provided us with the impetus
to probe the reasons responsible for such effects. In fact, the effect
of surface characteristics on the growth of peptide nanofilaments
1
0
has been reported recently.
It appears that the mesh-like structure for unsubstituted DKP 1
results from a discrete molecular cross-linked network derived
from a hydrogen bonding-mediated assembly. DKP 2 has a
lopsided structure which contains a puckered proline ring and an
aromatic indole skeleton. It has been proposed that 2 exists in
two different conformations, in which the diketopiperazine ring
exists in a typical boat conformation. DKP 3 possesses two
aromatic groups suggesting that p–p stacking is a dominant factor
behind self-organization. Such a possibility is further confirmed in
the solid state structure of 3 which exhibits aromatic interactions
(
Gly-Gly) 1, cyclo-(Trp-Pro) 2, cyclo-(Trp-Trp) 3, and cyclo-(Phe-
Phe) 4 (Fig. 1), and an investigation of the gross morphology of
self-assembled structures on different surfaces.9
Interestingly, atomic force microscopy (AFM) investigation of
the four samples (1-4; 0.3 mM in 50% aqueous methanol) afforded
diverse morphologies when studied on highly ordered pyrolytic
graphite (HOPG) surface. A fresh sample of 1 displayed a cross-
linked mesh-like network with a pore-size of 50–250 nm ( Fig.
9
c
2
a). In contrast, compound 2 lacked an ordered structure, while 3
displayed the formation of fibers and 4 exhibited a tape-like mor-
phology (Fig. 2b–d). DKPs 3 and 4 afforded similar morphology on a
more hydrophilic mica surface where we observed fibrous net-
(
van der Waals, hydrophobic, and electrostatic forces) between
the indole rings. Akin to nanotubular structures formed by
Phe-Phe, DKP 4 also reveals the formation of tubular structures,
possibly due to favorable aromatic interactions between the two
phenyl rings, which is further aided by the hydrophobic nature
of the HOPG surface.
*
Corresponding author. Tel.: +91 512 259 7643; fax: +91 512 259 7436.
E-mail address: sverma@iitk.ac.in (S. Verma).
0
040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.04.156

4
232
K. B. Joshi, S. Verma / Tetrahedron Letters 49 (2008) 4231–4234
Figure 2. AFM micrographs of fresh samples in 50% aqueous methanol of the self-assembled morphologies of compounds (a) 1, (b) 2, (c) 3, and (d) 4 on HOPG surfaces.
Micrographs of (e) 3 and (f) 4 on mica surfaces.
Encouraged by these results, we decided to further confirm the
structure of peptide aggregates by scanning electron microscopy
Fluorinated solvents are well-known for stabilizing peptide and
1
1
protein structures. The fiber cross-sections of fresh 3 and 4 in
50% aqueous methanol were ꢀ30 and ꢀ80 nm, respectively,
whereas in 50% aqueous 2,2,2-trifluoroethanol (TFE), the fiber
cross-section increased up to ꢀ500 and 200 nm for 3 and 4, respec-
tively, suggesting that the fluorinated solvent induces coalescence
in the aggregated ensembles. Interestingly, the structures observed
in 50% aqueous methanol were also retained in this solvent giving
rise to fiber-like aggregates on the HOPG surface (Fig. 4), suggest-
ing that the overall morphologies displayed by 3 and 4 remain
unaffected by a change in solvent composition.
(
SEM). Interestingly, SEM micrographs confirmed the AFM obser-
vations (Fig. 3a and b). A fresh solution of sample 1 in 50% aqueous
methanol demonstrated very dense sheet-like fibers (data not
shown).
Disordered and unsystematic aggregates of ꢀ1 lm diameter
were obtained for a fresh solution of compound 2 (data not
shown). A fresh solution of compound 3 afforded fibrillar morphol-
ogy, with diameter of 100–300 nm (Fig. 3a).
On the other hand, a solution of compound 4 in 50% aqueous
methanol showed bundled, straight fibers having diameters of
FT-IR measurements were used to probe significant structural
1
2
0
.4–1 lm (Fig. 3b). Aging of the solutions of samples 3 and 4 for
features responsible for the self-assembly process. The deconvo-
luted FT-IR spectra of the amide I and II regions of dipeptides 1, 2,
3, and 4, are shown in Figure 5. The amide I region of compound 1
2
days at ambient temperature resulted in mature fiber formation
(
Fig. S1; Supplementary data) confirming a time-dependent aggre-
gation and fibrillation event. A course fibrillar network was ob-
served after aging sample for days (Fig. S1a and c;
À1
contains three absorptions at 1619, 1678, and 1511 cm . The
À1
3
2
bands at 1678 and 1619 cm are usually assigned to anti-parallel
1
2a
À1
Supplementary data). However, the aged sample of 4, showed
bundled fiber formation which build up from the packing of small
fibers (Fig. S1b and d; Supplementary data).
b-sheets,
while the band at 1511 cm is representative of the
We further decided to determine the effect of solvent composi-
tion on the ultrastructures of the DKP ensembles formed in 3 and 4.
Figure 3. SEM micrographs of fresh samples of self-assembled morphologies of
compounds (a) 3 and (b) 4 in 50% aqueous methanol.
Figure 4. AFM micrographs of samples (a) 3 and (b) 4 in 50% aqueous TFE.

K. B. Joshi, S. Verma / Tetrahedron Letters 49 (2008) 4231–4234
4233
Figure 5. FT-IR deconvolution spectra. Amide I and II regions of compounds 1, 2, 3, and 4.
À1
presence of a b-sheet conformation. The bands at 1734 cm are
assigned to C@O stretching vibrations of the free or non-hydrogen
bonded groups, while that at 1705 cm is due to the bifurcated
presence of small amount of b conformation was also confirmed
À1
by a band at 1532 cm for compound 3. Compound 4 displayed
À1
À1
a band at 1742 cm due to the C@O stretching vibrations of free
À1
hydrogen bond or laterally hydrogen-bonded groups.
or non-hydrogen bonded groups, while a band at 1666 cm may
The amide I region of compound 2 exhibited bands at 1684 and
be attributed to b-turns with the participation of b-sheet confor-
mations.
À1
12a,13a–d
1
615 cm due to a b-sheet conformation with the measured com-
À1
ponents at 1645 cm
showing some structural randomness in
DKP supramolecular structures are governed primarily through
amide functionalities where they serve as interacting links via
hydrogen bonding interactions to reveal numerous structures such
as capsules, spheres, channels, helices, ribbons or tapes, rods, sheets
structures. Such structures were further confirmed by a band at
À1
1
543 cm in the amide II region which could tentatively be as-
À1
signed to an unordered component. The bands at 1746 cm were
1
3e–h
assigned to C@O stretching vibrations of free or non-hydrogen
or layers, and tubes.
The structural implications for the four
À1
bonded groups. However, the presence of a band at 1668 cm
DKPs 1–4, on the basis of FT-IR spectra, are summarized in Table 1.
The self-assembled structures were further evaluated to ascer-
tain their thermal stability, with the help of thermogravimetric
indicates the occurrence of b-turns together with a b-sheet confor-
À1
mation, as represented by a band at 1618 cm . Furthermore, the
Table 1
Structural implications of DKPs 1–4
DKP
DKP
cyclo-(Gly-Gly) 1
cyclo-(Trp-Pro) 2
cyclo-(Trp-Trp) 3
cyclo-(Phe-Phe) 4
À1
Frequency of the amide I band in cm
Deconvoluted bands
Secondary structures
1678, 1619
b-Sheets
1684, 1645, 1615
Random structures with b-sheets
1668, 1618
b-Turn and b-sheets
1666, 1614
b-Turn and b-sheets
Figure 6. TGA thermograms of (a) DKP 3 (black trace) and DKP 4 fiber (red trace); (b) derivative plots suggesting the higher stability of DKP 3 compared to DKP 4.

4
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K. B. Joshi, S. Verma / Tetrahedron Letters 49 (2008) 4231–4234
Figure 7. Proposed model for the fiber formation by compound 4 in solution state. (a) A single molecule of cyclo-(Phe-Phe), 4, (b) crystal packing extending along the ‘a’ axis,
and (c) an AFM micrograph of 4.
analysis (TGA), by monitoring the loss of weight as a function of
increasing temperature for compounds 3 and 4. Such studies have
been reported previously for peptide-based soft structures in order
to ascertain their thermal stability.14 The TGA thermograms of 3
and 4 showed ꢀ2–10% weight-loss going from room temperature
to ꢀ300 °C (Fig. 6a). A major decrease in weight was observed
above 300 °C for both 3 and 4 suggesting rugged thermal stability
of these DKPs perhaps due to the presence of aromatic substituents
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9
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morphology as observed by atomic force microscopy (Fig. 7).
In conclusion, this study gives an insight into possible contribu-
tions of aromatic amino acid side chains in DKP self-assembly and
allows us to propose that beneficial aromatic interactions might
dictate formation and stability of diketopiperazine ensembles. It
is expected that careful use of such interactions will help us to
understand their role as a stabilizing feature in aggregation and
as an aid in de novo design of self-assembled structures from pep-
tide-based molecular frameworks.
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1
1
K.B.J. thanks IIT-Kanpur for a graduate fellowship. This work is
supported by a Swarnajayanti Fellowship to S.V. from the Depart-
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Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2008.04.156.
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