Steric effect on the self-assembly behaviours of amino acid derivatives

Article abstract of DOI:10.1039/c4ra06582d

To investigate the steric effect of amino acids, N,N′-dialanine-1,4,5,8-naphthalene tetracarboxylic acid diethylamide (ANTA) and N,N′-diphenylalanine-1,4,5,8-naphthalene tetracarboxylic acid diethylamide (PNTA) were synthesized based on naphthalene-1,4,5,

Full text of DOI:10.1039/c4ra06582d

RSC Advances
View Article Online
View Journal | View Issue
PAPER
Steric effect on the self-assembly behaviours of
amino acid derivatives†
Cite this: RSC Adv., 2014, 4, 52245
Yulan Fan,‡ Linxiu Cheng,‡ Chunhua Liu, Yunzhi Xie, Wei Liu, Yongdong Li, Xun Li,
*
*
Yibao Li and Xiaolin Fan
To investigate the steric effect of amino acids, N,N0-dialanine-1,4,5,8-naphthalene tetracarboxylic acid
diethylamide (ANTA) and N,N0-diphenylalanine-1,4,5,8-naphthalene tetracarboxylic acid diethylamide
(PNTA) were synthesized based on naphthalene-1,4,5,8-tetracarboxylic dianhydride (NTA) with alanine
and phenylalanine, respectively. These compounds were characterized by infrared spectroscopy (IR),
magnetic resonance spectroscopy (1H NMR, 13C NMR) and mass spectrometry (MS). Interestingly, ANTA
could self-assemble into spiral nanorings, but PNTA formed nanospheres. In order to reveal the self-
assembly driving forces, the two-dimensional (2D) supramolecular self-assembly behaviours were
studied on highly oriented pyrolytic graphite (HOPG) by scanning tunneling microscopy (STM) at the
solid–liquid interface, which indicated the driving forces could be attributed to intermolecular hydrogen-
bonding and p–p stacking interactions. These results represent different self-assembly behaviours due
to the steric effect of amino acids.
Received 3rd July 2014
Accepted 29th September 2014
DOI: 10.1039/c4ra06582d
www.rsc.org/advances
an important role in the system of life. Therefore, amino acids
are very popular in the study of molecular recognition.13,14
1. Introduction
There are more than 20 kinds of amino acids in nature.
Therefore, thousands of structure-specic and different func-
tional peptides can be obtained through molecular design and
peptide synthesis. It is benecial for primitive selection of
peptides self-assembly and optimization of the self-assembled
condition. Based on the abovementioned advantages, in
recent years, domestic and foreign researchers have had
growing interest in self-assembling peptides. In 1993, Ghadiri
Supramolecular chemistry is an emerging discipline based on
non-covalent intermolecular interactions (known as weak
interactions) and the formation of complex and orderly
molecular aggregates.1 It is a cross-disciplinary eld, including
biology, chemistry, and materials science. Molecular recogni-
tion and supramolecular self-assembly are two very important
core concepts. The supramolecular self-assembly process is
characteristically spontaneous, occurring to endpoint without
any inuence of external forces.2 This is derived from the weak
intermolecular interactions, including hydrogen bonding,3 van
der Waals interaction,4 metal–ligand coordination,5,6 dipole–
dipole interaction,7 and p–p stacking interactions.8,9 Molecular
recognition was originally proposed with the aim to study
chemical problems of biological systems on the molecular level,
and it was related to specic intermolecular non-covalent bond
synergistic interactions. Molecular recognition is only con-
cerned about the weak interactions between a few molecules,
which widely exist not only among the biosphere (such as the
specic recognition between enzyme and substrate), but have
been used to open up new methods of synthetic chemistry.10–12
As the basic unit of macromolecular protein, amino acids play
et al.15 had designed and synthesized
a cyclic peptide
(cyclo-[(L-Gln-D-Ala-L-Glu-D-Ala) 2-]) containing 8 amino acid
residues. By alternating the spatial conformation (L- and D-) of
the amino acid residues, the cyclic peptide can be self-
assembled into nanotube structures in aqueous solution. In
the same year, Zhang et al.16 reported a complementary ionic
hexadecapeptide that can self-assemble into a kind of hydrogel
membrane. Subsequently, a series of peptides with self-
assembly behaviours have been reported.17–21
Peptide self-assembling can be divided into spontaneous
self-assembly and triggered self-assembly. Spontaneous self-
assembly means that the assembly can be formed spontane-
ously aer the peptide is dissolved in an aqueous solution. For
example, Zhang et al. reported the ionic complementary peptide
RAD series,16 which can self-assemble into nanotubes and
vesicles.22 The peptide RAD contains alternately arranged argi-
nine (R), aspartic acid (D) and alanine (A) residues and lipo-
some small peptides. Peptide-triggered self-assembly is a self-
assembly, which has been guided by changing the external
environment such as temperature, pH, and ion concentration.
Key Laboratory of Organo-pharmaceutical Chemistry, Gannan Normal University,
Ganzhou 341000, P. R. China. E-mail: liyb@gnnu.cn; fanxl2013@gnnu.cn; Fax: +86
07978393536
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c4ra06582d
‡ These authors contributed equally to this work.
This journal is © The Royal Society of Chemistry 2014
RSC Adv., 2014, 4, 52245–52249 | 52245

View Article Online
RSC Advances
Paper
Currently, most of the peptide self-assembling studies have
focused on triggered self-assembly for such self-assembly
structures with reversible and well controlled characteristics.
The previously reported peptide-triggered self-assembly
includes thermo-,23 pH-,24 photos-,25 ligand–receptor sensi-
tivity26 and other stimuli-responsive behaviors. Either sponta-
neous or triggered self-assembly depends on the secondary
structures such as the formation of a-helix, b-sheet or random
coil. However, it remains a challenge to prepare the structurally
controlled bionanomaterials with complex amino acids.
In this paper, we introduce a strong p–p conjugate nucleus
as the main driving force for self-assembly. Both ends of the
nucleus are modied amino acids aimed to investigate steric
effects. The self-assembly morphologies were observed by
atomic force microscopy (AFM). To further investigate the
interaction mechanism, the 2D self-assembly behaviours were
studied on HOPG substrate by STM.
2.4 Characterization
UV absorption spectra were obtained using a UV-2700 UV-Vis
spectrophotometer. The solutions were prepared by dissolving
both ANTA and PNTA into ethanol at room temperature. AFM
measurements were performed by using a Nanoscope IIIa
(Bruker, Germany). Absolute ethanol was used as the solvent to
prepare ANTA and PNTA with different concentrations. Molec-
ular self-assembly was accomplished using a pipette to drop
dilute solution droplets onto a freshly cleaved mica surface and
allowing the solvent to evaporate in air at room temperature.
STM measurements were performed using a Nanoscope IIIa
instrument (Bruker, Germany) under ambient conditions. All
STM images presented were recorded in constant current mode
using a mechanically cut Pt/Ir (80/20) tip. The sample with the
concentration (1.0 ꢂ 10ꢁ5 M) was prepared. Aer a droplet
(0.4 mL) of the solution was deposited onto a freshly cleaved
HOPG surface, the STM investigation was performed.
2. Experimental section
2.1 Materials
3. Results and discussion
The UV-Vis spectra in Fig. 1 showed that both ANTA and PNTA
generated two new absorption peaks at 358 nm and 378 nm,
respectively. Compared with the raw material NTA absorption
peak at 348 nm, the generation of a red-shied absorption peak
in the higher wavelength region indicated J-aggregate formation
in the self-assembled clusters.
Amino acids might spontaneously assemble into a variety of
structures. Under the same concentration condition, the micro-
aggregate structures of the dilute solutions of ANTA and PNTA
are discussed in detail.
It is interesting that many rings were observed at the mica
surface from the large AFM image in Fig. 2a which shows that
many nanorings are present on the mica surface with a dilute
solution of ANTA. In addition, it should be noted that the
nanoring presented obvious helical properties, as observed
from the magnied AFM phase image (Fig. 2b). In Fig. 2b, these
helix structures of ANTA could be observed distinctly in the AFM
phase image. The height prole of the structure indicates that
the average height and average diameter are about 19 nm and
490 nm, respectively (Fig. 2c and d). Therefore, the height of the
nanoring is much higher than a single ANTA molecule, which is
composed of many molecules. This interesting structure is due
to p–p stacking and hydrogen bonding interactions between
ANTA molecules.
All starting materials were purchased from commercial
suppliers and used without any further processing.
2.2 Synthesis of ANTA
0.2682 g (1 mmol) of naphthalene-1,4,5,8-tetracarboxylic dia-
nhydride, 0.1782 g (2 mmol) alanine and 2.0 g of imidazole were
heated at 120 ꢀC for 6 h under nitrogen atmosphere. Then 100
mL of ethanol was poured into the hot mixture, reuxed for 6 h
and kept overnight to allow precipitate formation. The resulting
light yellow precipitate was concentrated with decompression
lters aer being acidied with dilute HCl, then washed with
deionized water to neutral, ltrated and dried at 80 ꢀC under a
vacuum oven to yield 1.56 g light yellow powder (yield: 85%)
(Scheme 1). IR (KBr): 1579.6 cmꢁ1 (C]C), 1671.3, 1708.3 cmꢁ1
(C]O), 2941.2 cmꢁ1 (C–H), 3414.0 cmꢁ1 (O–H). 1H NMR
(DMSO, 400 MHz, ppm): 8.68–8.71 (s, 4H), 5.57–5.62 (q, 2H),
1.55–1.57 (d, 6H). 13C NMR (DMSO, 400 MHz, ppm): 171.43,
162.49, 131.37, 126.62, 49.46, 14.82.
MS (MALDI-TOF): 409.9 (calcd. 410.1, M).
2.3 Synthesis of PNTA
0.2682 g (1 mmol) of naphthalene-1,4,5,8-tetracarboxylic dia-
nhydride, 0.3303 g (2 mmol) phenylalanine and 2.0 g of imid-
azole were heated at 120 ꢀC for 6 h under nitrogen atmosphere.
In a similar procedure as the preparation of ANTA, a light yellow
powder was obtained (yield: 80%) (Scheme 1). IR (KBr) 1337.5,
1450.7, 1496.3, 1579.9 cmꢁ1 (C]C), 1705.1, 1958.3 cmꢁ1
(C]O), 2937.2 cmꢁ1 (C–H), 3028.0 cmꢁ1 (O–H). 1H NMR
(DMSO, 400 MHz, ppm): 8.58–8.69 (s, 4H), 7.15–7.16 (d, 4H),
7.08–7.12 (t, 4H), 7.02–7.06 (t, 2H), 3.59–3.61 (d, 1H), 3.56–3.57
(d, 1H), 3.27 (s, 2H). 13C NMR (DMSO, 400 MHz, ppm): 170.62,
162.46, 138.23, 131.71, 129.41, 128.61, 126.81, 126.48, 126.12,
55.00, 34.73.
The AFM image of PNTA in Fig. 3a shows small solid spheres
deposited on the mica surface, with an average height of about
5.6 nm. From the enlarged AFM image in Fig. 3b,
a
nanonecklace-like structure can be clearly observed, and it
MS (MALDI-TOF): 562.2 (calcd. 562.4, M).
Scheme 1 Synthesis route of ANTA and PNTA.
52246 | RSC Adv., 2014, 4, 52245–52249
This journal is © The Royal Society of Chemistry 2014

View Article Online
Paper
RSC Advances
Fig. 1 Ultraviolet-visible spectra of NTA, ANTA and PNTA in ethanol.
Fig. 3 Tapping mode AFM images of PNTA molecule on mica surface:
(a) AFM phase image of PNTA, the image size is 1.0 ꢂ 1.0 mm2, (b) AFM
height image of PNTA, the image size is 1.0 ꢂ 1.0 mm2; (c) height profile
of the section chosen in (b).
To further investigate the formation mechanism, the 2D self-
assembly behaviours were studied on HOPG substrate by STM.
Upon introduction of ANTA solution onto the HOPG surface, it
Fig. 2 Tapping mode AFM images of ANTA molecules on mica
surface: (a) large-scale AFM height image of ANTA, the image size is
10.0 ꢂ 10.0 mm2; (b) magnified AFM phase image of ANTA, the image
size is 1.0 ꢂ 1.0 mm2; (c) magnified AFM height image of ANTA, the image
size is 1.0 ꢂ 0.6 mm2; (d) height profile of the section chosen in (c).
consists of small solid spheres. The benzyl groups in the PNTA
molecules are relatively larger than the carboxyl groups, and the
steric effect of benzene rings impacts the formation of the
hydrogen bonding interactions between carboxyl groups.
The process of self-assembly is illustrated in Fig. 4. As shown
in Fig. 4a, ANTA molecules worked as both hydrogen acceptors
and hydrogen donors, which can bind with each other on the
basis of hydrogen-bonding interactions, and then assemble
spontaneously, based on p–p interactions, to successfully form
nanorings. However, in Fig. 4b, PNTA molecules were used as
building blocks via the introduction of p–p interactions, which
provided a guideline for the self-assembly behaviour, to
construct small solid spheres such as to form nanonecklace-like
structures. This is on account of the steric effect of the benzene
rings in PNTA, which restrain the generation of hydrogen
bonds.
Fig. 4 Schematic illustration of the formation of nanostructures: (a) of
ANTA; (b) of PNTA.
This journal is © The Royal Society of Chemistry 2014
RSC Adv., 2014, 4, 52245–52249 | 52247

View Article Online
RSC Advances
Paper
could be readily observed that ANTA self-assembled into 2D stacking interactions. The steric effect of amino acid derivatives
ordered monolayer structures. Fig. 5a showed a STM image on became potential factors for biomaterials building block
the graphite surface aer adsorption of ANTA in octanoic acid. because of special morphologies.
In this image, each molecule of ANTA was shaped like a rect-
angle with bright spots. In the channel between two connected
molecules, it presented slightly reduced contrast. Owing to the
Acknowledgements
higher electronic density of states of the aromatic rings, the This work was supported by National Natural Science Founda-
naphthalene of ANTA appeared in higher contrast than the tion of China (nos 21303024, 21365003), The Jiangxi Provincial
alanine residues in the image. The bright rectangle was attrib- “Ganpo Talents 555 Projects”, and Jiangxi Provincial Education
uted to the naphthalene core, whereas the surrounding line Department Fund (KJLD13080, GJJ13664), and the fundation of
with reduced contrast was assigned to the alanine residue. Innovation Team in Gannan Normal University (31260206) is
Careful analysis also revealed that the distance between two also gratefully acknowledged.
molecules was about 0.25 nm, which is suitable for hydrogen
bonding. The length of the bright part was measured to be 0.8 ꢃ
Notes and references
0.1 nm, which is consistent with the length of the backbone. On
the basis of this observation, a unit cell was marked in Fig. 5a.
According to this unit cell, four molecules are included in each unit
cell, and the parameters were measured to be a ¼ 1.0 ꢃ 0.2 nm,
b ¼ 1.7 ꢃ 0.2 nm and a ¼ 92 ꢃ 1.0ꢀ, respectively. According to
the STM images, the corresponding molecular model was
proposed in Fig. 5b. The molecules of adjacent lamellae adopt
head-to-tail packing geometry, which could be ascribed to the
hydrogen bond formation between carboxyl groups from
alanine residues. The p–p stacking interactions between ANTA
and substrate also played an important role in the self-
assembling process.
The STM results indicated that ANTA molecules could be
adsorbed at the liquid/solid interface and assembled into
ordered chain-like structures by hydrogen-bonding interactions
and p–p stacking interactions, while PNTA molecules showed
disordered arrangements. It is illustrated that the phenyl of
PNTA in the side group caused steric hindrance, which reduced
the hydrogen-bonding interactions and p–p stacking
interactions.
1 H. Ringsdolf, B. Schlaeb and J. Venzmer, Angew. Chem., Int.
Ed., 1988, 27, 113–158.
2 G. M. Whitesides, J. P. Mathias and C. T. Seto, Science, 1991,
254, 1312–1319.
3 M. Lackinger, S. Griessl, T. Markert and F. Jamitzky, J. Phys.
Chem. B, 2004, 108, 13652–13655.
4 M. Suzuki, Y. Nakajima, M. Yumoto, M. Kimura, H. Shirai
and K. Hanabusa, Langmuir, 2003, 19, 8622–8624.
5 A. Langner, S. L. Tait, N. Lin, C. Rajadurai, M. Ruben and
K. Kern, Proc. Natl. Acad. Sci. U. S. A., 2007, 104, 17927–17930.
6 A. Cnossen, D. Pijper, T. Kudernac, M. M. Pollard,
N. Katsonis and B. L. Feringa, Chem.–Eur. J., 2009, 15,
2768–2772.
¨
7 H. Spillmann, A. Kiebele, M. Stohr, T. A. Jung, D. Bonifazi,
F. Y. Cheng and F. A. Diederich, Adv. Mater., 2006, 18, 275–
279.
8 T. Kameda, S. Shimamori and T. Yoshioka, J. Phys. Chem.
Solids, 2012, 73, 120–123.
9 X. L. Wang, J. X. Zhang, G. C. Liu and H. Y. Lin, J. Solid State
Chem., 2011, 184, 280–288.
10 J. M. Lehn, Angew. Chem., Int. Ed., 1988, 27, 89–112.
11 D. J. Cram, Angew. Chem., Int. Ed., 1988, 27, 1009–1020.
12 C. J. Pedersen, Angew. Chem., Int. Ed., 1988, 27, 1021–1027.
13 C. A. Schmuck, Synlett, 2011, 13, 1798–1815.
14 J. H. Ha, J. M. Karchin, W. K. Nancy, L. S. Huang, E. A. Berry
and S. N. Loh, J. Mol. Biol., 2012, 416, 495–502.
15 M. R. Ghadiri, J. R. Granja, R. A. Milligan, D. E. Mcree and
N. Khazanovich, Nature, 1993, 366, 324–327.
16 S. G. Zhang, T. C. Holmes, C. Lockshin and A. Rich, Proc.
Natl. Acad. Sci. U. S. A., 1993, 90, 3334–3338.
17 X. H. Yan, Q. He, K. W. Wang, L. Duan, Y. Cui and J. B. Li,
Angew. Chem., 2007, 119, 2483–2486.
4. Conclusions
The steric effect of amino acids derivatives was investigated
mainly by AFM and STM. The results showed that the ANTA and
PNTA could self-assemble into nanorings and nanospheres,
respectively. According to STM results, the driving forces were
attributed to the intermolecular hydrogen bonds and p–p
18 R. J. Mart, R. D. Osborne, M. M. Stevens and R. V. Ulijn, So
Matter, 2006, 2, 822–835.
19 M. O. Guler and S. I. Stupp, J. Am. Chem. Soc., 2007, 129,
12082–12083.
20 M. Zelzer and R. Ulijin, Chem. Soc. Rev., 2010, 39, 3351–3357.
21 H. G. Cui, T. Muroaka, A. G. Cheetham and S. I. Stup, Nano
Lett., 2009, 9, 945–951.
22 S. Vauthey, S. Santoso, H. Y. Gong, N. Watsonand and
S. G. Zhang, Proc. Natl. Acad. Sci. U. S. A., 2002, 99, 5355–
5360.
Fig. 5 STM images of ANTA molecules on HOPG: (a) (14.63 ꢂ 14.63
nm2, Iset ¼ 1.2 nA, Vbias ¼ 159.9 mV) showing high-resolution STM
image of ANTA, (b) the tentative 2D packing model of ANTA molecules.
52248 | RSC Adv., 2014, 4, 52245–52249
This journal is © The Royal Society of Chemistry 2014

View Article Online
Paper
RSC Advances
23 B. Ciani, E. G. Hutchinson, R. B. Sessions and 25 L. A. Haines, K. Rajagopal, B. Ozbas, D. A. Salick,
D. N. Woolfson, J. Biol. Chem., 2002, 277, 10150–10155.
24 M. G. Ryadnov, B. Ceyhan, C. M. Niemeyer and
D. N. Woolfson, J. Am. Chem. Soc., 2003, 125, 9388–9394.
D. J. Pochan and J. P. Schneider, J. Am. Chem. Soc., 2005,
127, 17025–17029.
26 Y. Zhang, H. W. Gu, Z. M. Yang and B. Xu, J. Am. Chem. Soc.,
2003, 125, 13680–13681.
This journal is © The Royal Society of Chemistry 2014
RSC Adv., 2014, 4, 52245–52249 | 52249