Self-assembling nanotubes consisting of rigid cyclic γ-peptides

Article abstract of DOI:10.1002/adfm.201200488

Self-assembling cyclic peptide nanotubes (SPNs) have been extensively studied due to their potential applications in biology and material sciences. Cyclic γ-peptides, which have a larger conformational space, have received less attention than the cyclic α- and β-peptides. The self-assembly of cyclic homo-γ-tetrapeptide based on cis-3-aminocyclohexanecarboxylic acid (γ-Ach) residues, which can be easily synthesized by a one-pot process is investigated. Fourier transform infrared (FTIR) and NMR analysis along with density functional theory (DFT) calculations indicate that the cyclic homo-γ-tetrapeptide, with a non-planar conformation, can self-assemble into nanotubes through hydrogen-bond-mediated parallel stacking. Atomic force microscopy (AFM) and transmission electron microscopy (TEM) experiments reveal the formation of bundles of nanotubes in CH₂Cl₂/hexane, but individual nanotubes and bundles of only two nanotubes are obtained in water. The integration of TEG (triethylene glycol) monomethyl ether chains and cyclopeptide backbones may allow the control of width of single nanotubes.

Full text of DOI:10.1002/adfm.201200488

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Self-Assembling Nanotubes Consisting of Rigid Cyclic
γ-Peptides
Liangchun Li, Hongmei Zhan, Pengfei Duan, Jian Liao, Junming Quan, Yu Hu,
Zhongzhu Chen, Jin Zhu, Minghua Liu, Yun-Dong Wu,* and Jingen Deng*
in areas such as biology and materials sci-
ence.[3–6] In contrast to the morphologies
found in inorganic nanostructures, the
Self-assembling cyclic peptide nanotubes (SPNs) have been extensively
studied due to their potential applications in biology and material sci-
ences. Cyclic γ-peptides, which have a larger conformational space, have
received less attention than the cyclic α- and β-peptides. The self-assembly
of cyclic homo-γ-tetrapeptide based on cis-3-aminocyclohexanecarboxylic
acid (γ-Ach) residues, which can be easily synthesized by a one-pot process
is investigated. Fourier transform infrared (FTIR) and NMR analysis along
with density functional theory (DFT) calculations indicate that the cyclic
homo-γ-tetrapeptide, with a non-planar conformation, can self-assemble into
nanotubes through hydrogen-bond-mediated parallel stacking. Atomic force
microscopy (AFM) and transmission electron microscopy (TEM) experiments
reveal the formation of bundles of nanotubes in CH2Cl2/hexane, but indi-
vidual nanotubes and bundles of only two nanotubes are obtained in water.
The integration of TEG (triethylene glycol) monomethyl ether chains and
cyclopeptide backbones may allow the control of width of single nanotubes.
nanotubes formed by cyclopeptide aggre-
gate into bundles[3–9] that may have appli-
cations in the preparation of nanosized
wires.[7,10] Recently, it was reported that the
self-assembling morphologies could be con-
trolled by polymer-conjugated peptides.[11,12]
The majority of cyclic peptide nano-
tubes so far have hydrophilic inner sur-
faces.[3] Granja et. al. designed and syn-
thesized cyclic α,γ-peptides, which formed
tubular structures via antiparallel β-sheet-
like hydrogen bonds.[7,13–15] In such struc-
tures, the β-methylene moiety of cis-3-
aminocyclohexanecarboxylic acid (γ-Ach)
residues is projected into the lumen of the
cylinder, creating a partially hydrophobic
cavity, which has different transport/car-
rier properties in polar/aploar solvents
with the modified pore interior.[13–15] However, the investigation
of the self-assembling morphologies of cyclic homo-γ-peptides
has not been reported. Herein, we report the first preparation
of cyclic γ-peptide 2 (Scheme 1) by one-pot cyclodimerization
of dipeptide 1, which has a hydrophobic inner surface that is
1. Introduction
Novel functional nanomaterials based on self-organizing systems
are of great current interest.[1,2] Self-assembling peptide nanotubes
(SPNs), formed from cyclic oligopeptides consisting of various
amino acids, are widely studied for their potential applications
Dr. L. C. Li, Prof. J. G. Deng
Key Laboratory of Drug-Targeting of Education
Ministry and Department of Medicinal Chemistry
West China School of Pharmacy
Sichuan University
Chengdu 610041, China
E-mail: jgdeng@cioc.ac.cn
H. M. Zhan, Prof. J. Liao, Y. Hu, Z. Chen, Prof. J. Zhu
Key Laboratory of Asymmetric Synthesis & Chirotechnology
of Sichuan Province
Chengdu Institute of Organic Chemistry the Chinese Academy of Sciences
Chengdu 610041, China
P. F. Duan, Prof. M. H. Liu
Institute of Chemistry
the Chinese Academy of Sciences
Beijing, 100080, China
Dr. J. M. Quan, Prof. Y.-D. Wu
School of Chemical Biology and Biotechnology
Peking University Shenzhen Graduate School
Shenzhen 518055, China
E-mail: chydwu@ust.hk
Scheme 1. Synthesis of 2 : a) H2, Pd-C (10%), DCM; b) TFA, DCM;
DOI: 10.1002/adfm.201200488
c) HATU, HOAt, DIPEA, DCM.
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knowledge, this is the first example of cyclic peptides bearing
EG side chains.[19,20]
2. Results and Discussion
Cyclic tetrapeptide 2a was conveniently prepared from the
dimerization of deprotected dipeptide that was resulted from
hetero-dipeptide 1a and obtained in 42% yield by column chro-
matography with CHCl3 and MeOH (30:1, v/v) as mobile phase
(Scheme 1). The structure of 2a was confirmed by NMR and
ESI-HRMS analyses. Moreover, the clean formation of 2a was
indicated by examining the crude product using MALDI-TOF
mass, which revealed [M+Na]+ peak with a mass/charge ratio
of 1412. The fact that no other cyclopeptides and oligopeptides
were detected by MALDI-TOF suggested that the rigid confor-
mation of γ-Ach residues had a high propensity for forming the
cyclic tetrapeptide.
1H NMR spectroscopy was then employed to probe the
1
aggregation of 2a in solution. In DMSO-d6, H NMR spectrum
of 2a displayed sharp and well-resolved signals (Figure 1a).
NOESY spectrum of 2a in DMSO-d6 (Figure S10, Supporting
Information) shows that the N-H and Cγ-H bonds were trans to
each other while the N-H and the Cα-H of adjacent cyclohexane
rings were in a cis relationship, as shown in Figure 2a,b.
While in CDCl3 solution, poor-resolved peaks with significant
line-broadening that showed concentration-dependence were
Figure 1. Selected region of 1H NMR spectrum of a solution of 2a: a) in
DMSO, b) 10 mM, c) 20 mM, and d) 40 mM are in CDCl3, showing the
downfield shifts of two Ph-H and N-H.
decorated by four methylene groups contributed by the cyclo-
alkane rings. Moreover, we demonstrate that well dispersed
individual nanotubes can be formed from
the cyclic peptide bearing dendritic PEG side
chains through parallel β-sheet-like stacking.
Cyclic γ-peptides, which have a larger con-
formational space, have received less atten-
tion than the cyclic α- and β-peptides.[3] For
the design of cyclic γ-peptide nanotubes,
γ-Ach was chosen as the basic residue,
because the cyclohexane ring of γ-Ach is
expected to stabilize the desired flat con-
formation of cyclic γ-peptide. Such a flat
conformation should favor self-assembly of
the cyclic γ-peptide into tubular structures
through face-to-face molecular stacking
mediated by multiple hydrogen bonds.[13–16]
Thus, cyclic homo-γ-peptide 2 was designed
to consist of alternating chirality of (1R,3S)-
γ-Ach and (1S,3R)-γ-Ach residues. The latter
residue carries a hydroxyl group that allows
the attachment of various functional groups,
which not only increases the solubility of the
peptide, but also allows to modify the property
of the nanomaterials formed by the peptide
for applications in areas such as biosensing,
pharmaceuticals, catalysis and electronics,
etc.[1–6] It is well known that PEGylation leads
to improved biocompatibility of proteins and
reduced biodegradation rates.[17] Based on our
own experience,[18] 3,5-bis(triethylene glycol
Figure 2. Theoretical and calculated dimer resulting from a,c) parallel and b,d) antiparallel
β-sheet-like hydrogen bonding of cyclic peptides. The side chains are shown as R for simplified
model and the geometry of dimers (all nonpolar hydrogen atoms were merged for clarity) were
optimized with B3LYP/6-31G(d) and shown as a top view (top) and side view (bottom).
monomethyl ether)benzoate, containing two
tri-EG (TEG) monomethyl ethers, was used
to modify (1S,3R)-γ-Ach. To the best of our
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observed (Figure 1b–d, Figure S6 (Supporting Information)).
The NH signals lying at >8.00 ppm (10–40 mM in CDCl3) sug-
gest that these protons are involved in hydrogen-bonding. Thus,
these observations are consistent with hydrogen-bond-mediated
aggregation of 2a in CDCl3, giving rise to multiple supra-
molecular species in fast exchange on the NMR time scale.[21]
The aromatic ring proton (Ph-H) signals change from 6.58 and
6.97 ppm (in Figure 1b) to 5.85 and 6.69 ppm (Figure 1d) upon
the increase of concentration from 10 mM to 40 mM. The sig-
nificant upfield shifts of these signals indicate that there is π–π
stacking interactions between adjacent monomers upon aggre-
gation. Furthermore, an NOE cross peak between the Ph-H and
the Cε-H of modified γ-Ach residues from adjacent cyclopep-
tide molecule exists in the NOESY in CDCl3 (Figure S11 vs
S10 (Supporting Information)). This suggests that the adjacent
cyclopeptide molecules stack in a parallel fashion.
In the gas phase the BSSE-corrected binding energies calcu-
lated with the M05-2X/6-31G+(d,p) method[25–27] are −34.4 and
−23.0 kcal/mol for the parallel and antiparallel dimers, respec-
tively. The parallel dimer is more stable than the antiparallel
one by about 11.4 kcal/mol, because the latter forms weaker
hydrogen bonds judging from hydrogen-bonding distances
and angles and also suffers from a geometrical deformation.
The solvent effect on the binding energy was estimated by
the IEFPCM (integral equation formalism of the polarizable
continuum model)[28,29] method at the M05-2X/6-31G+(d,p)
level using the gas phase optimized structures. The dimeri-
zation energy of the parallel dimer in chloroform is reduced
to −12.4 kcal/mol, while that of the antiparallel one becomes
0.1 kcal/mol. These results suggest that the parallel stacking of
2a is energetically much more favourable than the antiparallel
one, and the latter may not be stably formed.
Moreover, FT-IR studies of films deposited on KBr pellets
as well as in solution showed substantial evidence for the self-
assembly of the peptide by β-sheet like hydrogen bonding (see
Supporting Information). The FT-IR spectrum of 2a in CHCl3
showed an amide I absorption (C = O stretching mode), an
amide II absorption (mainly N-H bending and C-H stretching
modes) and an amide A absorption (N-H stretching mode) at
1633 cm−1 , 1552 cm−1 and 3286 cm−1 , respectively. The observed
amide A band indicates a tight intermolecular hydrogen
bonding, and the amide I and II absorptions are characteristic
for nanotubes formed from β-sheet-like networks.[13–15,22,23]
In the meantime, the absence of signal near 1690 cm−1 corre-
sponding to antiparallel β-sheet in 2a further suggests parallel
β-sheet structures in this compound.[22,23]
Parallel and antiparallel β-sheet-like hydrogen-bonding of
cyclic peptides can both explain the self-assembling behavior
(Figure 2a, 2b). To better understand the conformation of this
cyclic peptide, the structural and self-assembling features of
2a were explored by density functional theory (DFT)[24] calcu-
lations. Because of the conformational rigidity of the γ-Ach
residues, only one low-energy conformation was found for the
backbone of 2a and it is noteworthy that the four cyclohexanes
form a non-planar (boat-shaped with side view) conformation
with an S4 symmetry (Figure S17, S18, Supporting Informa-
tion). Further calculations showed that 2a could form a reason-
able dimer via parallel or antiparallel β-sheet-like hydrogen
bonding (Figure 2c, 2d and Figure S19, S20 (Supporting
Information)). When the optimized structures of the dimers
are viewed along the dimer axes (top view in Figures 2c and
2d), it is apparent that one of the monomers is slightly rotated
with respect to the other, and the monomers of the antiparallel
stacking are rotated more than the parallel stacking. This fea-
ture may be attributed to the geometrical characteristic of C =
O···HN hydrogen bonds (the best alignment would be 120°
for C = O···H angles due to the directionality of the oxygen
lone pairs) and steric repulsions. Furthermore, the non-
planar conformation of 2a is more fitted for parallel stacking
because of the better alignment of the structures. The dis-
tance between Ph-H and Cε-H of modified γ-Ach residue from
adjacent cyclopeptide molecules in the parallel stacking is
shorter (3.23 Å) than that in the antiparallel stacking (7.00 Å)
(Figure S12, Supporting Information), which is in agreement
with the NOESY experiment.
Thus, the above NMR, FT-IR experiments and DFT calcu-
lations all suggest the formation of tightly hydrogen-bonded
parallel β-sheet structures for cyclopeptide 2a in CDCl3. We
have also obtained more direct evidence for the conforma-
tional insights. Previous experiments of cyclopeptides indicated
that partial N-methylation of the NH groups of one face in
this cyclic γ-peptide may be an excellent strategy for the study
of stacking mode.[6,13–15,21] Unfortunately, the preparation of
N-methyl cyclopeptide of 2a is unsuccessful. Thus, N-methyl
cyclopeptide 2b was designed and synthesized to study the self-
assembly properties (Scheme 1). As shown in Figure 3a, the 1H
NMR spectrum of 2b in CDCl3 is well defined, which reflects
a C2 symmetry of the monomeric species, and the NH signal
at 5.60 ppm shows that it is not involved in hydrogen-bonding.
These indicate that 2b cannot dimerize through antiparallel
stacking (parallel stacking is prohibited by the methylation),
which is in qualitative agreement with the previously mentioned
DFT calculations. When equimolecular 2b and its enantiomer
were mixed in CDCl3, a broad NH signal at 7.50–7.80 ppm of
1H NMR spectrum appeared (Figure 3b), indicating the forma-
tion of hydrogen-bonds between the two enantiomers of 2b.
In this case, parallel hydrogen-bonding (Figure 4) is possible.
As shown in Figure 5, an NOE cross peak between theγ-H
of N-methylated amino acid (MeN-γ-Ach) and the NH of the
other γ-Ach exists in the NOESY (can only be observed in the
assembly, Figure 4), which strongly suggest the parallel dimer
structure. This is in an excellent agreement with the previous
observations for the aggregation of 2a. It can be concluded that
cyclotetrapeptide 2a self-assemble via parallel stacking instead
of antiparallel stacking to form nanotubes.
a
*
b
**
*
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
f1 (ppm)
Figure 3. Selected region of 1H NMR spectrum of a) enantiomerically
pure peptide 2b in CDCl3 (30 mM) and b) a mixture of peptides 2b and
its enantiomer (20 mM). ∗ denotes the NH signal in the monomer
(5.60 ppm), ∗∗ denotes the NH signal in the dimer (7.60 ppm).
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O
Me
N
O
N
H
4.0
4.5
Me
N
O
RO
OR
N
Cγ-H of MeN-γ-Ach
5.0
OR
H
O
5.5
O
H
N-H of monomer
6.0
6.5
7.0
7.5
8.0
8.5
9.0
N
O
N
H
O
Me
N
N
RO
Me
N-H of dimer
O
Figure 4. Theoretical heterodimer. The side chains are shown as R for
simplified model. The structure results from parallel β-sheet-like hydrogen
bonding of cyclic peptides 2b and its enantiomer.
9.0
8.5
8.0
7.5
7.0
6.5
f2 (ppm)
6.0
5.5
5.0
4.5
4.0
Figure 5. Selected region of the NOESY spectrum of equimolecular 2b
and its entantiomer in CDCl3.
A solution of 2a in CH2Cl2 (2 mM) was equilibrated against
n-hexane via vapor-phase diffusion. After incubating at the
room temperature for three days it led to the formation of a
colorless gel. The organogel was then dispersed in n-hexane by
sonication. The resulting sample
with height of 1.4-1.6 nm (Figure 6b,d), same as the molecular
dimension of the cyclic peptide 2a. More detailed informa-
tion of the sample was obtained from transmission electron
was examined using atom force
(a)
(b)
(e)
microscopy (AFM). A high reso-
lution AFM image of the gel
dispersed in n-hexane showed a
fiber with a height of 2.1–7.8 nm
and a length over several hun-
dred nanometres (Figure 6a,c).
Self-assembling in aqueous
media is of particular interest.
Recently, dendritic aromatic
amphiphiles bearing branched
EG chains were shown inter-
(c)
nm
(d)
esting property of enhanced self-
assembly into a variety of nano-
nm
8
structures in aqueous media.
1.4
7
One-dimensional tubular aggre-
6
gates were also formed by the
self-assembly of an amphiphilic
5
0.9
0.4
4
rigid macrocycle in aqueous
3
solution.[30] The self-assembling
2
behaviour of 2a, which has a
1
rigid hydrophobic interior and
0
0
flexible hydrophilic TEG exterior
nm
46
0
22
nm
226
0
56
112
171
sidechains, was investigated in
aqueous media. A direct investi-
gation on the aggregation of 2a
in aqueous solution was difficult
because of its poor solubility in
water. Thus, the organogel of 2a
formed in CH2Cl2/n-hexane was
dispersed in water by sonication.
A drop of the dispersed suspen-
sion was then placed on a mica
surface and dried in vacuum.
The AFM image of the material
after being treated with water
revealed long filaments (>1 μm)
(f)
Figure 6. a,b) AFM images of the gels dispersed in n-hexane and in water; c,d) profile obtained along the
black line shown in (a,b), respectively. e) TEM field of negatively stained filaments. f) Schematic illustration
of branched TEG-peptide nanotubes adsorbed onto a mica surface.
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multiplet (m) or broad (br). Carbon nuclear magnetic resonance (13C
NMR) spectra were recorded on Bruker WM-600 MHz or WM-300 MHz
spectrometers. Electrospray (ESI) mass spectra were recorded on a
Bruker BioTOF Q mass spectrum. FT-IR measurements were made on
a Nicolet MX-1E spectrometer by the KBr method or the 40 mM CHCl3
solution FT-IR measurements placed in a 0.1 mm KBr solution IR cell
were made on a ThermoFisher Nicolet 6700 spectrometer.
microscopy (TEM), which revealed the presence of long fila-
ments (>400 nm) with widths of ∼5 nm and ∼9 nm (Figure 6e,
shown by black and white arrows, respectively). Thus the AFM
and TEM images reveal that the nanomaterial of 2a dispersed in
water exists in both individual nanotubes as shown in Figure 6f
(height of about 1.5 nm and width of about 5 nm) and bundles
of only two nanotubes (width of about 9 nm). These results also
suggest that the TEG side chains of a nanotube extended onto
the surface of mica.[11,12]
It is quite interesting that the bundled nanotubes of 2a
formed in CH2Cl2/n-hexane deassemble into mono nano-
tubes and bundles of only two nanotubes upon dispersing
into water. It is likely that the hydrophilic TEG chains play a
key role. When they are sufficiently hydrated, they suppress lat-
eral aggregation.[11,12,19,20,30] Meanwhile, the hydrophobic inter-
actions among the cyclohexyl groups, π–π stacking interactions
between the benzene rings, and the intermolecular hydrogen
bonds are strong enough to maintain the nanotubes in aqueous
media. It is worth noting that, in Granja’s mixed α,γ-cyclic pep-
tide system the 1D nanotubes[7] formed via antiparallel-sheet
hydrogen-bonding interaction and the interstrand salt-bridge
interactions, while in our system the 1D nanotubes formed via
parallel-sheet hydrogen-bonding interaction.
Synthesis of CP 2a: After two vacuum/H2 cycles to replace air inside
the reaction tube with hydrogen, the mixture of 1a (50 mg, 0.06 mmol),
10% Pd/C (10 wt% of the substrate) in DCM (2 mL) was vigorously
stirred at room temperature (ca. 20 °C) under 1 atm of hydrogen for
24 h. The reaction mixture was filtered using silica gel, and the filtrate
was concentrated. Then the BOC group of the residue was removed by
trifluoroacetic acid (TFA)/DCM (1:1, v/v), and the product was dissolved
in dry 30 mL of DCM. To the solution, HOAt (9 mg, 0.07 mmol), HATU
(28 mg, 0.07 mmol), and N,N-Diisopropylethylamine (DIPEA) (40 μL,
0.25 mmol) were added at room temperature. After stirring the resulting
solution until completion of the reaction, the solution was concentrated
in vacuum. 50 mL of CHCl3 was added to the residue and stirred, filtered,
and then the combined organic filtrates were washed with 10% KHSO4
and 10% Na2CO3. After the organic layer was dried over anhydrous
Na2SO4 and concentrated in vacuum, the crude cyclopeptide was purified
by column chromatography with CHCl3 and MeOH (30:1, v/v) as mobile
1
phase to provide a pure 2a (35 mg) in 42% yield. H NMR (600 MHz,
CDCl3+CD3OD, δ): 0.89-0.95 (m, 4H), 1.11-1.26 (m, 6H), 1.35-1.40
(q, J = 11.8 Hz, 2H), 1.44-1.46(m, 2H), 1.61(d, J = 12.0 Hz, 2H), 1.67-
1.76(m, 8H), 1.81-1.83(m, 2H), 2.02(d, J = 12.0 Hz, 2H), 2.11-2.19 (m,
4H), 2.27(t, J = 12.0 Hz, 2H), 3.30 (s, 12H), 3.48-3.49 (m, 8H), 3.58-
3.62 (m, 18H), 3.65-3.67(m, 8H), 3.78-3.83(m, 10H), 4.07 (t, J = 4.4 Hz,
8H), 4.87-4.92 (m, 2H), 6.61 (t, J = 2.2 Hz, 2H), 7.10 (d, J = 2.1 Hz,
4H), 7.47 (d, J = 7.8 Hz, 4H); 13C NMR (75 MHz, CDCl3+CD3OD, δ):
23.4, 25.4, 29.5, 30.4, 31.3, 35.9, 37.4, 38.2, 39.8, 42.9, 45.2, 47.2, 58.7,
67.6, 69.4, 70.3, 70.4, 70.6, 71.2, 71.7, 106.5, 108.0, 131.8, 159.6, 165.5,
174.5, 176.5; FT-IR (KBr): ν = 3283.3 (amide A), 2926.2, 1717.7, 1640.2
(amide I), 1596.4, 1547.6 (amide II), 1447.0, 1296.7, 1176.7, 1103.7,
851.8, 762.0; FT-IR (40 mM, 293 K, CHCl3): ν = 3286.0 (amide A), 2935.3,
1633.2 (amide I), 1600.1, 1552.2 (amide II), 1446.1, 1299.5, 1241.5,
1217.2, 1175.0, 1104.0; HRMS (ESI, m/z): calcd. for C70H108N4Na2O24
([M + 2Na]2+): 717.3569, found: 717.3589; MALDI-TOF (m/z): 1412.0
[M+Na] +.
3. Conclusions
In summary, we have investigated the self-assembly of cyclic γ-
peptide 2a, which can be easily synthesized by a one-pot process.
FT-IR and NMR analysis along with DFT calculations indicate
that 2a, with a non-planar conformation, can self-assemble into
nanotubes through hydrogen-bond-mediated parallel stacking.
AFM and TEM experiments reveal the formation of bundles of
nanotubes in CH2Cl2/n-hexane; but individual nanotubes and
bundles of only two nanotubes are obtained in water. The inte-
gration of TEG chains and cyclopeptide backbones may allow
the control of width of single nanotubes. Further investigation
on these issues is currently being carried out to better under-
stand the conformation of analogous γ-peptides and potential
transport/carrier applications.
Synthesis of CP 2b: 2b was synthesized using the same method like
1
2a but starting from 1b and yield in 30%. H NMR (600 MHz, CDCl3,
δ): 0.84-0.97 (m, 2H), 1.25-1.35 (m, 4H), 1.37-1.48 (m, 6H), 1.52-1.58
(m, 2H), 1.66-1.73 (m, 12H), 1.80-1.89 (m, 8H), 1.91-1.95 (m, 4H), 2.08
(d, J = 12.5 Hz, 2H), 2.18 (t, J = 12.1 Hz, 2H), 2.32 (d, J = 11.0 Hz, 2H),
2.79-2.83(m, 2H), 2.87 (s, 6H), 4.01 (t, J = 6.6 Hz, 8H), 4.05-4.08 (m,
2H), 4.44 (t, J = 11.8 Hz, 2H),5.01-5.05 (m, 2H),5.60 (d, J = 9.2 Hz, 2H),
6.64 (s, 2H), 7.13(d, J = 2.2 Hz, 4H); 13C NMR (150 MHz, CDCl3, δ):
22.6, 23.9, 25.0, 25.9, 28.4, 29.7, 32.4, 34.6, 35.7, 36.4, 37.0, 37.9, 44.4,
45.2, 66.7, 51.9, 70.9, 106.4, 107.8, 132.0, 160.2, 165.6, 173.9, 175.0;
HRMS (ESI, m/z): calcd. for C64H96N4O12Na ([M + Na]+): 1135.6922,
found: 1135.6973.
4. Experimental Section
General: γ-Ach and modified γ-Ach were prepared according to previous
reports.[31,32]
O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
Preparation of the Gels: A solution of 2 mM of 2a in CH2Cl2 equilibrated
against n-hexane via vapor-phase diffusion, and then incubated at room
temperature for three days, the colorless gel was formed.
hexafluorophosphate (HATU), 1-Hydroxy-7-azabenzotriazole (HOAt)
were used as obtained from GL Biochem (Shanghai) Ltd. All other
reagents obtained from commercial suppliers were used without further
purification unless otherwise noted. Dichloromethane (DCM) was dried
and distilled over calcium hydride. Analytical thin-layer chromatography
was performed on silica gel GF254 plates from Qingdao Haiyang Chemical
Co. Ltd. Silica gel flash chromatography was performed using silica
gel (200–300 mesh) from Qingdao Haiyang Chemical Co. Ltd. Proton
nuclear magnetic resonance (1H NMR) spectra were recorded on Bruker
WM-600 MHz or WM-300 MHz spectrometers. Chemical shifts were
reported in parts per million (ppm, δ ) from TMS or solvent resonance
AFM Measurements: After the gels were dispersed in n-hexane by
sonication, a piece of clean hydrophobic silicon slide was dipped into
the hot n-hexane solution of the gels and cooled to room temperature,
then the slide was dried under vacuum for AFM measurement. Similarly,
after the gels were dispersed in water by sonication, a drop of dispersed
suspension was placed on a mica surface and dried in vacuum for
AFM measurement. All AFM pictures were measured with a Digital
Instrument Nanoscope III Multimode system (Santa; Barbara, CA)
with a silicon cantilever using the tapping mode. All the AFM images
are shown in the height mode without any image processing except
flattening.
1
as the internal standard. H NMR splitting patterns are designated as
singlet (s), doublet (d), triplet (t), quartet (q). All first-order splitting
patterns were assigned on the basis of the appearance of the multiplet.
Splitting patterns that could not be easily interpreted are designated as
TEM Measurements: The gels were dispersed in water by sonication
and droplets of 10 μL of the suspension were placed onto the specimen
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Adv. Funct. Mater. 2012,
DOI: 10.1002/adfm.201200488
2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
5

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©
6
wileyonlinelibrary.com
2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Funct. Mater. 2012,
DOI: 10.1002/adfm.201200488