The self-assembly of cystine-bridged γ-peptide-based cyclic peptide-dendron hybrids

Article abstract of DOI:10.1039/c3ob40532j

Novel cystine-bridged γ-peptide-based cyclic peptide-dendron hybrids have been synthesized by oxidative coupling between two cysteine residues of the linear peptides via the formation of disulfide bonds in high yields. The self-assembly of the hybrids was

Full text of DOI:10.1039/c3ob40532j

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PAPER
The self-assembly of cystine-bridged γ-peptide-based
cyclic peptide–dendron hybrids†
Cite this: Org. Biomol. Chem., 2013, 11,
8443
Zhizhong Lin,a,d Liangchun Li,*c Yujin Yang,a Hongmei Zhan,a Yu Hu,a
Zhiming Zhou,c Jin Zhu,a Qiwei Wanga and Jingen Deng*b,e
Novel cystine-bridged γ-peptide-based cyclic peptide–dendron hybrids have been synthesized by oxi-
dative coupling between two cysteine residues of the linear peptides via the formation of disulfide
bonds in high yields. The self-assembly of the hybrids was studied by FT-IR, 1H NMR, TEM, and AFM ana-
lyses which indicate that the nanotube was constructed through intermolecular hydrogen-bonding of
the hydrophobic cyclic peptide moieties and possesses amphiphilic property by conjugating a hydrophilic
dendron on the exterior of the cyclic peptide ring. The diameters of nanofibers that consisted of nano-
tubes depend on the employed solvent in the self-assembly process, and uniform filaments formed from
double amphiphilic nanotubes via hydrophobic interactions between their hydrophobic faces have been
observed in water as well as in aqueous solutions.
Received 16th March 2013,
Accepted 22nd October 2013
DOI: 10.1039/c3ob40532j
www.rsc.org/obc
the nanotube interior. Granja and co-workers12–15 developed
α,γ-cyclic peptide nanotubes consisting of alternating
1 Introduction
Currently there is intense interest in the formation of nano- D-α-amino acids and (1R,3S)-3-aminocyclohexanecarboxylic
structures with technological or biomedical applications based acids (γ-Achs)19 in which the C2 methylene group of each cyclo-
on their self-assembly through the spontaneous interactions of hexyl moiety is projected into the lumen of the cylinder, gener-
their component molecules.1,2 Cyclic peptides (CPs), due to ating a partially hydrophobic cavity. The α,γ-CPNs favor the
their efficiency in limiting the conformational flexibility and encapsulation of apolar solvents,12,20,21 which shows potential
ease in modulation of their three-dimensional (3D) structures, applications in drug delivery.17,22,23
are one of the extensively studied self-assembly building
The main challenge in designing a self-assembling fibrous
blocks.2–5 To date, cyclic D,L-α-peptides,6,7 β-peptides,8–10 system is to control the assembly to form uniform reproduci-
δ-peptides,11 and the hybrids including α- and γ- or ε-amino ble structures. Recently, we reported that γ-CP composed of
acids12–16 have been applied to self-assemble into well-defined γ-Achs only self-assembled into γ-CPN, in which the projection
nanotubes. The majority of cyclic peptide nanotubes (CPNs) so of C2 methylene of all γ-Achs into the lumen creates a more
far have hydrophilic inner surfaces and can only be permeated hydrophobic interior in the organic nanotubes.24 Moreover,
by polar molecules.3,7,9,17 To further control the transport pro- the dendritic side-chains can modulate the physical properties
perties of a wide range of molecules through self-assembling to improve the solubility of the γ-CP and control the aggrega-
peptide nanotubes (SPNs), artificial amino acids containing tion of the CPNs in solvents, and also promote the stability of
rigid cycloalkanes,12–14 aromatic rings,16,18 and unsaturated the CPN due to the aromatic π–π stacking interactions between
groups11 have been used to modify the physical properties of adjacent dendrons upon association of the γ-CPs.24,25 The
side-chain functional groups of the cyclic peptide have an
influence on the formation of either single-peptide nanotubes
or higher-order 3D aggregates.15,18,24,26 Peptide–dendron27,28
and peptide–polymer4,29–32 hybrids offer the potential to study
how the conformational properties of two folded structural
elements all allosterically communicate structural infor-
mation. On the other hand, the self-assembly of amphiphilic
components leading to microphase separation constitutes a
powerful approach toward the fabrication of complex supramo-
lecular architectures, especially tubular nanostructures due
to both the hydrophobic and hydrophilic interactions.27,28,33
Furthermore, for the applications in biomedicine and
aChengdu Institute of Organic Chemistry, Chinese Academy of Sciences,
Chengdu 610041, China
bKey Laboratory of Drug-Targeting of Education Ministry, West China School of
Pharmacy, Sichuan University, Chengdu 610041, China. E-mail: jgdeng@scu.edu.cn
cSchool of Life Science and Engineering, Southwest University of Science and
Technology, Mianyang, 621010, China. E-mail: lilc76@gmail.com
dUniversity of Chinese Academy of Sciences, Beijing, 100049, China
eJiangxi Key Laboratory of Organic Chemistry, Jiangxi Science & Technology Normal
University, Nanchang 330013, China
†Electronic supplementary information (ESI) available: Scheme S1, Fig. S1–S22,
complete experimental procedures, and characterization of cyclic peptide–
dendron hybrids and spectral data. See DOI: 10.1039/c3ob40532j
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biomaterials, it is desirable to be able to tailor pore sizes and peptides, benzoates with branched Tg (triethylene glycol
pore surfaces of CPs. In our preliminary experiments, we were monomethyl ether) chains (Fig. 1) were chosen to modify the
unsuccessful in synthesizing γ-CPs with larger pore diameter, γ-CPs.24 The synthesis of 1a and 1b, which are conjugated with
such as cyclic γ-hexapeptides composed of homochiral or alter- the first- and second-generation hydrophilic dendrons respect-
nating chiral γ-Achs, although N-methylated cyclic γ-hexa- ively, will be performed by coupling two cysteine residues of
peptides have been obtained.34,35 It was reported that the linear peptides on the basis of the formation of disulfide
macropeptides have been conveniently prepared by the for- bonds.36,37 Amphiphilic nanotubes can be formed from the
mation of a disulfide bond between two cysteine residues,36,37 self-assembly of 1a and 1b via β-sheet-like hydrogen-
and the self-organization of cystine-based macrocycles facili- bond-mediated parallel stacking, respectively, on which a
tated the formation of the tubular nanostructures.38–40 Herein, hydrophobic wall (both the inner and outer surfaces are
we would like to describe the synthesis and self-assembly be- hydrophobic) is constructed by the self-organization of γ-tetra-
havior of novel disulfide-bond containing CP-dendron hybrids peptide moieties bearing hydrophobic cyclohexane back-
(Fig. 1, 1a and 1b) composed of a homochiral γ-tetrapeptide bones12,21,24 (Fig. 2). Therefore, in an aqueous environment,
and one bridged cystine residue attached to hydrophilic den- the hydrophobic side of a single tube is completely buried
dritic side-chains on the exterior of the CP-ring.24,34
against other hydrophobic tube faces to aggregate into
In our previous research, we found that a linear tetra- bundles of CPNs due to the hydrophobic interactions, around
peptide consisting of only chiral γ-Achs has very poor solubility which the hydrophilic dendritic side-chains like to project
in common solvents. For increasing the solubility of the toward the aqueous media because of hydrate. We investigate
the impact of different assembly methods and solvents on the
overall nanoscale dimensions (i.e., length and width), and we
show that the diameters of nanofibers depend on the employed
solvent in the self-assembly process. To the best of our knowl-
edge, there has been no report that the aggregation of CPNs can
be controlled using their amphiphilic characteristics.1–4,41,42
2 Results and discussion
The synthetic strategy is outlined in Scheme 1. Initially, for the
synthesis of 1a and 1b,43 the first- and second-generation den-
dritic benzoic acids were attached to the amino group of
methyl L-cysteinate 2 respectively and subsequent deprotection
Fig. 1 The structures of 1a and 1b.
was performed by hydrolysis of the methyl esters. (1R,3S)-Ach
Fig. 2 Schematic representation of the hybrids 1 and their supramolecular stacking mediated by intermolecular hydrogen-bonding, shown by dotted lines.
Cartoon representation showing the self-assembly of the hybrids 1 into amphiphilic nanotubes and then into nanofibers.
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Scheme 1 Synthesis of 1. (a) EDCI, HOBt, DIPEA, CH2Cl2; (b) LiOH, THF–H2O–CH3OH; (c) HCl (gas), CH2Cl2; (d) I2, CH3OH–CH2Cl2.
was prepared according to our method.19 Boc-protected 4 and corresponding mass signals for different ion adducts.43 As
methyl protected 5 were prepared with the corresponding stan- expected, the second-generation dendron conjugated cyclic
dard methods. Dipeptide 6 was obtained by coupling between hexapeptide 1b has better solubility than the first-generation
acid 4 (Boc-γ-Ach-OH) and amine 5 (H-γ-Ach-OMe) with good one 1a in common solvents such as CHCl3 and CH3CN, as well
yield. In the presence of EDCI, HOBt and DIPEA, the depro- as aqueous solution.
tected dipeptides 7 and 8 were coupled respectively with corres-
The rod-like assemblies of 1a were gained from (CHCl3–
pondingly modified cysteines 2 and 3, giving the tripeptides 9 CH3OH) via vapor-phase diffusion (see ESI, Fig. S2†), but they
and 10. The linear hexapeptides 11 that consisted of one are unsuitable for single-crystal X-ray diffraction. Fourier trans-
cysteine residue grafted with the Tg-dendrons at the N-termi- form infrared (FT-IR) spectroscopy of 1a in the solid state
nus, another cysteine residue at the C-terminus, and a γ-tetra- (Fig. S3†) shows a sharp peak at 1631.8 cm−1 in the amide I
peptide constructed from four (1R,3S)-Ach residues in the region, and 1535.8 cm−1 in the amide II region, which are
middle were synthesized by coupling of 9 and 10 after respect- characteristic of extensively hydrogen-bonding β-sheet-like
ive deprotection. The cyclization of linear hexapeptides 11 pro- networks.44–46 Moreover, the N–H stretching frequency occurring
ceeded by simple iodine oxidation between the cysteine at 3305.3 cm−1 also corresponds to tightly hydrogen bonded
residues, where side chains were protected with trityl groups, ring-stacked networks. These results suggest that the needle-
constructing desired disulfide bonds in one step, including shaped fibers are constructed from tightly packed nanotubes
both deprotection and oxidative coupling to lead to 1a and 1b formed by self-assembly of 1a on the basis of β-sheet-like hydro-
in 73% and 49% yields, respectively.36,37 The molecular gen-bonding. These observations also prompted us to investi-
structures of 1a and 1b were identified by NMR, IR, and gate the self-assembly behavior of 1a and 1b in the solutions.
HRMS analyses. Moreover, the formation of cyclic peptides
was also confirmed by MALDI-TOF-MS analysis showing the via vapor-phase diffusion against n-hexane, and colorless gels
A solution of 1a in CHCl3 (6 mg mL−1) was equilibrated
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Fig. 3 (a–c) TEM images of the gels formed by 1a and then dispersed in
n-hexane, water and water–ethanol (1 : 1, v/v), respectively; (d) AFM images of the
1a sample adsorbed on mica, showing nanofibers; (e) height profile measured
along the line shown in (d).
were formed at room temperature for two days.47 As the initial
study, the organogels were dispersed in n-hexane by soni-
cation. The resulting sample was dried on a carbon-coated
copper TEM grid, which was then negatively stained with a
phosphotungstic acid solution. Under these conditions, trans-
Fig. 4 (a–c) TEM images of the gels formed by 1b and then dispersed in
n-hexane, water and water–ethanol (1 : 1, v/v), respectively. (d, f) AFM images
of the gels of 1b dispersed in water and water–ethanol (1 : 1, v/v), showing
mission electron microscopy (TEM) revealed the presence of nanofibers; (e, g) height profile measured along the line shown in (d, f ).
long fibers with a width of about 100 nm (Fig. 3a). By disper-
sing the gels in pure water, a fibrous assembly of ca. 92 nm in
diameter was obtained (Fig. 3b). When the gels of 1a were samples were investigated by AFM and the AFM images indi-
dispersed in a mixture of water and ethanol (1 : 1, v/v), cated the formation of filaments with a uniform diameter and
TEM studies revealed the presence of filaments with widths of an average height of 2.06 nm (Fig. 4d–e and S5†) and 2.04 nm
about 8 nm (Fig. 3c). This dimension of the aggregate is (Fig. 4f–g and S6†) for the gels of 1b dispersed in water and
approximately twice the extended molecular length (4 0.5 nm water–ethanol (1 : 1, v/v) respectively, suggesting that the
in molecular length of 1a by the Corey–Pauling–Koltun (CPK) second-generation benzyl ether-dendron bearing hydrophilic
molecular model).33 The same sample was also determined by Tg-chains could impact the height of the filaments by compar-
atomic force microscopy (AFM) and the AFM image showed ing with the result of 1a (Fig. 2). Thus the TEM and AFM
the nanofiber with an average height of 1.75 nm that corres- images revealed that nanostructures of 1b dispersed in water–
ponds to the estimated width of a naked cyclic peptide of the ethanol (1 : 1, v/v) exist in both individual nanotubes and
hybrid 1 (1.7 0.1 nm by the CPK model) (Fig. 3d–e and S4†). bundles of only two nanotubes, but in water only fibrous
These results implied that the uniform filaments only consist bundles consisting of two nanotubes exist.
of two nanotubes.
Self-assembling in aqueous media is of particular interest
According to the same procedure, the gels of 1b were pre- for applications in biomedicine.1,41,48–51 Amphiphilic com-
pared and dispersed in n-hexane;47 TEM images showed long ponents, especially those bearing EG (ethylene glycol) chains,
ribbon-like aggregates with diameters of 140–300 nm (Fig. 4a). have shown an interesting property of enhanced self-assembly
When the gels were dispersed in pure water, in contrast to 1a into a variety of nanostructures in aqueous media.1,41 In this
that formed fibers with a large diameter, 1b showed cylindrical work, for improving the solubility of CPs, the hybrid 1b conju-
aggregates with diameters ranging from 8 nm to 10 nm gated with the second-generation dendritic Tg-chains was
(Fig. 4b). By considering the extended molecular lengths of 1b synthesized and shows good solubility in aqueous solution.24
(5 0.5 nm by the CPK model), the images indicate that the Thus, 1b was dissolved in acetonitrile, water, and their mixture
diameter of cylindrical aggregates corresponds to double the (water–CH3CN, 5 : 95; v/v) respectively by heating and the
molecular length. By dispersing the gels of 1b in water– resulting transparent solution was cooled to room temperature
ethanol (1 : 1, v/v), TEM images displayed discrete filaments of overnight. TEM studies were carried out on a copper grid.
about 6 nm and 10 nm in diameter, respectively (Fig. 4c When the self-assembly of 1b was performed in pure aceto-
respectively shows black and white arrows). Furthermore, the nitrile, visual formation of the hybrid assemblies occurred
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amphiphilic nanotubes because of solvation of Tg-side chains
to suppress lateral aggregation.24 It is interesting that only 1b
can be dispersed in water to form uniform filaments of double
amphiphilic nanotubes. In the polar CH3CN, only a single
nanotube self-assembled from the hybrid 1b was observed. In
pure water or even mixing a small amount of water (5%) into
the CH3CN phase, double tubular nanostructures were only
formed from 1b. However, the nanofibers became shorter with
the addition of water probably because of the competition by
water for CP–CP intermolecular hydrogen-bonding sites hin-
dering further organization of nanotubes.21,52 In aqueous solu-
Fig. 5 TEM images of the tubular nanostructures formed by the self-assembly
of 1b in CH3CN (a), 5% H2O in CH3CN (b), and H2O (c).
under these conditions. TEM images showed uniform fibrils tion, amphiphilic nanotubes especially self-assembled from 1b
with a diameter of about 5 nm and more than 430 nm in via β-sheet-like hydrogen-bonding were further organized from
length (Fig. 5a), which corresponded to an individual colum- individual nanotubes into double tubular nanostructures in
nar structure of the nanotube organized from 1b on the basis order to decrease the exposure of the hydrophobic faces of the
of β-sheet-like hydrogen-bonding (Fig. 2). In a mixture of 5% amphiphilic nanotubes to aqueous media. Two amphiphilic
water in CH3CN, fibrous assemblies of about 10 nm in dia- nanotubes possess an inherent tendency to hold together by
meter were observed and a maximum length could be roughly strong hydrophobic interactions between the hydrophobic faces
estimated with more than 110 nm (Fig. 5b). Notably, in pure of neighboring tubes in aqueous solution, and the sufficient
water, highly uniform filaments of 10 nm in diameter with a hydration of the dendritic Tg-chains attached to other faces of
shorter length (size ranging from 45 to 180 nm) were also the tubes suppresses the lateral aggregation (Fig. 2).
found by TEM analysis (Fig. 5c), which is in good agreement
with double size of the molecular length of 1b, suggesting that gation tendency of the hybrids 1 in solution. The spectrum of
the filaments consisted of only two nanotubes. 1a even in the polar solvent DMSO-d6 displays poor-resolved
1H NMR spectroscopy was then used to probe the aggre-
These results indicate that the hybrid 1 composed of a CP- signals (Fig. S13†), showing the strong intermolecular aggre-
ring bearing a hydrophobic γ-tetrapeptide moiety and a hydro- gation. Notably, in DMSO-d6, 1H NMR spectrum of 1b is sharp
philic dendritic pendant may self-assemble into well-ordered with well-resolved signals (Fig. 6a)43 and 2D NOESY analysis
and amphiphilic tubular nanostructures on the basis of inter- (Fig. S14 and S15†) shows that the N–H and Cα–H bonds of
molecular hydrogen-bonding of the CP-rings. Further aggre- adjacent cyclohexane rings as well as cysteine residue are in a
gation of the amphiphilic nanotubes depends on the cis relationship, as shown in Fig. 2 and Fig. S1.† Fortunately,
employed solvent system. In the nonpolar CHCl3–n-hexane in CDCl3, the spectrum of 1b shows peaks that broaden
system, both hybrids 1a and 1b formed a bundle of nano- obviously but also well-defined and significant concentration-
fibers, which can be dispersed in a mixture of water and ethanol dependent chemical shifts, suggesting that the N–H
(1 : 1, v/v) as uniform filaments consisting of two or single groups participate in hydrogen-bonding interaction for the
Fig. 6 Selected region of 1H NMR spectra of 1b (a) in DMSO-d6, (b) 2 mM, (c) 10 mM, (d) 20 mM, and (e) 40 mM in CDCl3, showing the downfield shifts of N–H
signals. The “*” sign denotes the Cα–H signal in cysteine residues (5.32 ppm).
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Fig. 7 Temperature dependence of the 1H NMR spectrum of 10 mM of 1b in CDCl3 (a, −10 °C; b, 0 °C; c, 10 °C; d, 20 °C; e, 30 °C; f, 40 °C; g, 50 °C), showing the
upfield shifts of N–H signals.
intermolecular organization, although the presence of slowly
interconverting conformers on the NMR time scale indicates
that there are more than six peaks for the N–H groups
(Fig. 6b–e). Moreover, the N–H signal assigned as the exocyclic
amide proton (NH(6), also see: Fig. 2) in the downfield
(8.24 ppm, in 2 mM solution in CDCl3) only displays 0.05 ppm
downfield shift on increasing the concentration to 40 mM,
which implies that the exocyclic amide formed a stable inter-
molecular hydrogen-bonding.13 1H NMR spectra of the hybrid
1b in CDCl3 at lower temperature show broad signals in the
amide proton region indicating that the hybrid 1b formed
intermolecular hydrogen-bonding (Fig. 7a and 7b). With the
elevation of temperature, these chemical shift values move to
upfield from 8.48 to 8.13 ppm and the signals become more
and more well-resolved, suggesting weakening of the hydro-
gen-bonding (Fig. 7).
Solution FT-IR investigations furnished additional evidence
of self-assembled β-sheet structures in the nanotubes (Fig. 8,
Table 1; also see: Fig. S7–12†).24,46,48,49,53 The observed N–H
stretching frequencies (amide A bands) of 3302 and 3301 cm−1
for 1a in CHCl3 and 1b in CH3CN respectively indicate tightly
intermolecular hydrogen-bonding. The FT-IR measurement
also shows amide I bands at 1636–1638 cm−1 and amide II
bands at 1537–1539 cm−1 for 1a in CHCl3, 1b in CH3CN and
Fig. 8 Selected region of FT-IR spectra of 1a (a) in CHCl3 and (b) in the gel
D2O that are characteristic of nanotubes formed from β-sheet-
like networks. In contrast to the result of FT-IR analysis for 1a
state and FT-IR spectra of 1b (c–f) in CHCl3, gel state, CH3CN and D2O,
respectively.
in CHCl3, the FT-IR spectrum of 1b in CHCl3 displays the
amide I band at 1649 cm−1 and amide II band at 1508 cm−1
(Fig. 8c, also see Table 1) that are not consistent with a typical
β-sheet structure, in which the band at 1649 cm−1 is fairly similar to aggregate in CHCl3, probably as a result of aromatic π–π
to the random coil peak54,55 (around 1654 cm−1) and the peak stacking interactions between the adjacent first-generation
at 1508 cm−1 is located in the regions of nonbonding amide II dendrons on the exterior of CP24 as well as from the intermole-
bands (1503–1516 cm−1) observed in the CHCl3 solutions.53 cular hydrogen-bonding between the exocyclic amides of 1a
The FT-IR analysis for 1a shows that there is a strong tendency (Fig. 2 and S1a†). However, the steric hindrance between the
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stacking between cyclic hexapeptides of the hybrids. TEM as
well as AFM measurements reveal that width-controllable
nanofibers consisting of a single or two nanotubes have been
obtained by self-assembling, and the diameter of nanofibers
depends on the employed solvent in the self-assembly process.
Especially, the hybrid 1b conjugated with the second-
generation benzyl ether-dendron bearing hydrophilic Tg-chains
possesses good solubility in common solvents and has self-
assembled into highly uniform nanofibers in polar solvents.
Shorter nanofibers were formed in water or aqueous aceto-
nitrile. These results indicate that the integration of dendritic
EG chains and hydrophobic cyclic peptides in the amphiphilic
nanotubes may allow the control of the width as well as length
of the nanofibers aggregated by β-sheet-like hydrogen-bonding
between the cyclic hexapeptides of the hybrids. Moreover,
the design of introducing disulfide-bonds into the cyclic
γ-peptides might permit control over the pore diameter simply
by adjusting the number of γ-Achs subunits.38 We believe that
uniform nanofibers have potential applications in nanotech-
nological fields such as drug delivery and catalysis. Further
investigation on this issue is currently being carried out to
better understand the conformation of analogous γ-peptides
and potential transport/carrier applications.
Table 1 Absorption frequencies of hybrids in different media
Frequencies [cm−1
]
Compounds
Solventsa
Amide Ab
Amide I
Amide II
1a
1b
CHCl3
Gels
CHCl3
Gels
CH3CN
D2O
3302
3302
3294 (3431)
3300
1636
1630
1649
1632
1638
1636
1537
1533
1508
1534
1538
1539
3301
c
a The concentrations of the samples in solutions were 10 mM for both
1a in CHCl3 and 1b in D2O. The concentrations of 1b in both CHCl3
and CH3CN were 20 mM. b Values in parentheses are for putative
amide bands arising from non-hydrogen-bonding NH. c It is impossible
to subtract the solvent absorption in this region because of the strong
ν (O–D) band of water.
second-generation dendritic Tg-chains conjugated on the
exterior of CP of 1b, especially after the solvation of Tg-chains
in CHCl3, may inhibit the association of 1b to form intermole-
cular hydrogen-bonds and thereby the β-sheet structures.30,32
The addition of n-hexane resulted in the decrease of the sol-
vation of the dendritic Tg-chains and hence triggers the aggre-
gation process to form organogels. Really, in contrast to the
CHCl3 solution, the FT-IR spectrum of 1b for the gels formed
in a mixture of CHCl3 and n-hexane exhibits the characteristic
amide I and II bands at 1632 and 1534 cm−1 respectively for
β-sheet hydrogen-bonding (Fig. 8d and Table 1). The CP-rings
of 1 can self-assemble by stacking on top of one another
through contiguous parallel or antiparallel β-sheet-like hydro-
gen-bonding on either side. As shown in Fig. 2 and Fig. S1,†
five inter-ring hydrogen-bonds might form between the cyclic
hexapeptides of 1 with an exocyclic hydrogen-bond between
the dendritic amides in the parallel model (Fig. 2 and S1a†),
while only four intermolecular hydrogen-bonds exist in the
antiparallel models (Fig. S1b–c†). Moreover, in the antiparallel
models, the exocyclic hydrogen-bonding cannot proceed due
to the γ-Ach residue shift of one unit toward the right or left.
These suggest that the parallel stacking of hybrids 1 is energe-
tically much more favorable than the antiparallel ones. In par-
ticular, this is supported by the fact that the exocyclic amide
proton (NH(6)) signal gradually upfield shifts on increasing
the temperature from −10 to 50 °C (Fig. 7), and the amide IA
absorptions at 1636–1638 cm−1, in the absence of the charac-
teristic amide IB bands at 1680–1695 cm−1, imply the formation
of a parallel β-sheet conformation in the nanotubes.24,46,53
4 Experimental
General
(1R,3S)-3-Aminocyclohexanecarboxylic acid (γ-Ach) was pre-
pared according to our method.19 O-(7-Azabenzotriazol-1-yl)-1,
1,3,3-tetramethyluronium hexafluorophosphate (HATU),
1-hydroxy-7-azabenzotriazole (HOAT), 1-hydroxybenzotriazole
(HOBt) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide
hydrochloride (EDCI) were all 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 chrom-
atography was performed on silica gel GF254 plates from
Qingdao Haiyang Chemical Co. Ltd. Silica gel flash chromato-
graphy was performed using silica gel (200–300 mesh) from
Qingdao Haiyang Chemical Co. Ltd. Melting points were
recorded on a BÜCHI Melting Point B-545 apparatus and are
uncorrected. Optical rotations were measured with an auto-
matic Perkin-Elmer 341 digital polarimeter; concentrations are
given in g per 100 mL. Proton nuclear magnetic resonance
(1H NMR) spectra were recorded on Bruker WM-300 MHz or
600 MHz spectrometers. Chemical shifts are reported in parts
per million (ppm, δ) from TMS or solvent resonance as the
3 Conclusions
In summary, we have studied the self-assembly of cystine- internal standard. 1H NMR splitting patterns are designated as
bridged γ-peptide-based cyclic peptide–dendron hybrids 1a singlet (s), doublet (d), triplet (t), and quartet (q). All first-
and 1b, which were conveniently synthesized by oxidative order splitting patterns were assigned on the basis of the
coupling between two cysteine residues of the linear peptides appearance of the multiplet. Splitting patterns that could not
on the basis of the formation of disulfide bonds in high yields. be easily interpreted are designated as multiplet (m) or
1H NMR and FT-IR analyses show that 1a and 1b can self- broad (br). Carbon nuclear magnetic resonance (13C NMR)
assemble by β-sheet-like hydrogen-bond-mediated parallel spectra were recorded on Bruker WM-300 MHz or 600 MHz
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spectrometers. Electrospray (ESI) mass spectra were recorded
on a Bruker BioTOF Q mass spectrum.
Method B: 1b was dissolved in acetonitrile or water or their
mixture (0.1 mg mL−1), and then heated in an oil bath at
Synthesis of cyclic peptide–dendron hybrids.43 Synthesis of 100 °C to yield a transparent solution. The water content in
1a: To a solution of I2 (101 mg, 0.4 mmol) in CH3OH–CH2Cl2 acetonitrile was 5% (v/v). The resulting solution of 1b was
(10 : 90, 120 mL), 11a (30 mg, 0.017 mmol) in CH3OH–CH2Cl2 cooled to room temperature overnight.
(10 : 90, 30 mL) was added dropwise at −10 °C, and the result-
Transmission electron microscopy (TEM). After the sample
ing mixture was stirred at −10 °C for 20 minutes. After 2 M for TEM was prepared, droplets of 10 µL of the solution were
Na2S2O3 (100 mL) was added, the reaction mixture was concen- placed on the specimen holder, a 200 mesh copper grid. After
trated. The solution was extracted with CHCl3 (3 × 30 mL). The 2 min, the grid was stained with 1% (w/v) phosphotungstic
combined organic layers were dried over MgSO4 and concen- acid for 1 min and the excess fluid was removed. Samples were
trated in vacuo. The residue was purified by column chromato- viewed using a HITACHI H-600 electron microscope for TEM at
graphy on silica gel (CHCl3–CH3OH = 30 : 1, v/v) to give 1a an accelerating voltage of 75 kV.
(16 mg, 73%) as a white solid. Mp: decomposed at 278 °C;
Atom force microscopy (AFM) measurements. After the gels
[α]2D0 = −12 (c = 0.2, CHCl3–MeOH = 3 : 1, v/v); FT-IR (ν/cm−1): were dispersed in water or a water–ethanol (1 : 1, v/v) mixture,
3443, 3296, 2932, 2859, 1747, 1634, 1582, 1546, 1496, 1452, 10 µL of the suspension was deposited on a freshly cleaved
1397, 1334, 1218, 1112; ESI-HRMS, calculated for piece of mica and left to adhere overnight. AFM imaging was
C63H102N6Na2O20S2 [M + 2Na]2+: 686.3913; found 686.3187; MS performed at room temperature using the tapping mode on a
(MALDI-TOF), calculated for C63H102N6NaO20S2 [M + Na]+: Seiko SPI3800N. According to the manufacturer, the probes
1349.6; found 1349.8.
used were etched silicon probes with a typical tip radius of
Synthesis of 1b: To a solution of I2 (594 mg, 2.34 mmol) in <10 nm; the normal spring constant was 3 N m−1. The drive
CH3OH–CH2Cl2 (10 : 90, 400 mL), 11b (251.3 mg, 0.1 mmol) in frequency was around 75 kHz.
CH3OH–CH2Cl2 (10 : 90, 100 mL) was added dropwise at
Solution FT-IR measurements. FT-IR measurements were
−10 °C, and the resulting mixture was stirred at −10 °C for performed with the CHCl3 and CH3CN solution placed in a
4 hours. After 2 M Na2S2O3 (100 mL) was added, the reaction 0.1 mm KBr solution IR cell, and the D2O solution was
mixture was concentrated to remove organic solvents. Then, measured in a CaF2 cell. All samples were recorded on a
the solution was extracted with CHCl3 (3 × 50 mL). The com- NICOLET 6700 FT-IR Spectrometer.
bined organic layers were dried over anhydrous Na2SO4 and
concentrated in vacuo. The residue was purified by column
chromatography on silica gel (CHCl3–CH3OH = 40 : 1, v/v) to
Acknowledgements
give 1b (99 mg, 49%) as a white solid. Mp: decomposed at
271 °C; [α]2D0 = −16 (c = 0.2, CH2Cl2); 1H NMR (DMSO-d6,
600 MHz, ppm), δ 1.23–1.35 (m, 16H), 1.64–1.91 (m, 16H),
2.07–2.37 (m, 4H), 2.99–3.16 (m, 4H), 3.20, 3.22 (2 × s, 18H),
3.38–3.42 (m, 12H), 3.48–3.58 (m, 27H), 3.65–3.72 (m, 15H),
4.00 (brs, 4H), 4.09 (brs, 8H), 4.56–4.66 (m, 2H), 5.00 (s, 4H),
6.77 (s, 4H), 6.83 (s, 1H), 7.16 (s, 2H), 7.42–7.43 (m, 2H, NH),
7.65 (br s, 1H, NH), 8.10 (d, 1H, NH, J = 6.5 Hz), 8.32 (d, 1H,
NH, J = 7.7 Hz), 8.56 (d, 1H, NH, J = 8.0 Hz); 13C NMR (DMSO-
d6, 150 MHz, ppm), δ 23.26, 24.08, 24.41, 28.16, 28.29, 30.88,
31.39, 31.74, 32.05, 35.59, 35.72, 36.13, 43.15, 44.23, 44.80,
47.47, 47.72, 48.34, 51.50, 52.53, 53.05, 58.49, 68.91, 69.49,
70.09, 70.21, 70.32, 70.45, 71.75, 72.28, 105.40, 107.06, 107.54,
132.47, 136.33, 137.76, 152.65, 159.83, 166.00, 168.96, 171.33,
173.76, 173.94, 174.37, 174.98; FT-IR (ν/cm−1): 3436, 3307,
3056, 2931, 2863, 1746, 1635, 1593, 1535, 1505, 1439, 1347,
1332, 1296, 1249, 1112, 941, 846; ESI-HRMS, calculated for
C98H156KN6O34S2 [M + K]+: 2064.0; found 2063.9; MS (MALDI-
TOF), calculated for C98H156NaN6O34S2 [M + Na]+: 2048.0;
found 2048.5.
We thank the National Natural Science Foundation of China
(grant no. 91027022, 21002099 and 20428202), the National
Basic Research Program of China (973 Program, no.
2010CB833300), and Sichuan University for financial support
of the research. The Chengdu Institute of Organic Chemistry,
Chinese Academy of Sciences, and West China School of Phar-
macy, Sichuan University, contributed equally to this work.
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