Chiral self-assembly of designed amphiphiles: Optimization for nanotube formation

Article abstract of DOI:10.1021/la3030606

Four amphiphiles with l-aspartic acid headgroups (Asp) and a diphenyldiazenyl group (Azo) contained within the hydrophobic tails were designed and synthesized for self-assembly into helically based nanotubes. The amphiphiles of the form R'-{4-[(4-alkylphenyl)diazenyl]phenoxy}alkanoyl-l- aspartic acid (where R' is 10 or 11) varied only in alkyl chain lengths either side of the azo group, having 4, 7, or 10 carbon distal chains and 10 or 11 carbon proximal chains (R-Azo-R'-Asp, where R denotes the number of carbons in the distal chain and R' denotes the number of carbons in the proximal chain). Despite the molecular similarities, distinct differences were identified in the chiral order of the structures self-assembled from hot methanolic aqueous solutions using microscopy and spectroscopic analyses. This was reflected in dominant thermodynamic aggregate morphologies that ranged from amorphous material for 10-Azo-10-Asp, through twisted ribbons (196 ± 49 nm pitch) for 7-Azo-11-Asp, to the desired helically based nanotubes for 4- and 7-Azo-10-Asp (81 ± 11 and 76 ± 6 nm diameters, respectively). Another key variable in the self-assembly of the amphiphiles was the use of a second method to precipitate aggregates from solution at room temperature. This method enabled the isolation of thermodynamically unstable and key transitional structures. Helical ribbons were precursor structures to the nanotubes formed from 4- and 7-Azo-10-Asp as well as the wide, flattened nanotube structures (587 ± 85 nm width) found for 4-Azo-10-Asp. Overall, the results highlighted the interplay of influence of the headgroup and the hydrophobic tail on self-assembly, providing a basis for future rational design of self-assembling amphiphiles.

Full text of DOI:10.1021/la3030606

Article
pubs.acs.org/Langmuir
Chiral Self-Assembly of Designed Amphiphiles: Optimization for
Nanotube Formation
Thomas G. Barclay,*,† Kristina Constantopoulos,† Wei Zhang,‡,§ Michiya Fujiki,‡ and Janis G. Matisons†,⊥
†Flinders Centre for Nanoscale Science & Technology, School of Chemical and Physical Sciences, Flinders University, Adelaide,
Australia
‡Graduate School of Materials Science, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan
S
* Supporting Information
ABSTRACT: Four amphiphiles with L-aspartic acid headgroups
(Asp) and a diphenyldiazenyl group (Azo) contained within the
hydrophobic tails were designed and synthesized for self-assembly
into helically based nanotubes. The amphiphiles of the form R′-
{4-[(4-alkylphenyl)diazenyl]phenoxy}alkanoyl-L-aspartic acid
(where R′ is 10 or 11) varied only in alkyl chain lengths either
side of the azo group, having 4, 7, or 10 carbon distal chains and
10 or 11 carbon proximal chains (R-Azo-R′-Asp, where R denotes
the number of carbons in the distal chain and R′ denotes the
number of carbons in the proximal chain). Despite the molecular
similarities, distinct differences were identified in the chiral order
of the structures self-assembled from hot methanolic aqueous
solutions using microscopy and spectroscopic analyses. This was reflected in dominant thermodynamic aggregate morphologies
that ranged from amorphous material for 10-Azo-10-Asp, through twisted ribbons (196 49 nm pitch) for 7-Azo-11-Asp, to the
desired helically based nanotubes for 4- and 7-Azo-10-Asp (81 11 and 76 6 nm diameters, respectively). Another key
variable in the self-assembly of the amphiphiles was the use of a second method to precipitate aggregates from solution at room
temperature. This method enabled the isolation of thermodynamically unstable and key transitional structures. Helical ribbons
were precursor structures to the nanotubes formed from 4- and 7-Azo-10-Asp as well as the wide, flattened nanotube structures
(587 85 nm width) found for 4-Azo-10-Asp. Overall, the results highlighted the interplay of influence of the headgroup and the
hydrophobic tail on self-assembly, providing a basis for future rational design of self-assembling amphiphiles.
directing parameters on the self-assembly of the desired
aggregates.
INTRODUCTION
■
A very useful structure in nanotechnology is the one-
dimensional container or nanotube. One type of nanotube
with flexible utility is constructed through the helical wrapping
of bilayer ribbons self-assembled from amphiphiles. Such
nanotubes generally have hydrophilic surfaces, hosting a large
range of different chemical functionalities, and also have an
array of internal diameters.1−3 This flexibility in chemistry and
size, along with their shape, provides a range of useful
applications including (i) encapsulating other substances for
subsequent controlled release, such as in drug delivery,4,5 (ii)
acting as templates for the organization of metals,6,7 semi-
conductors,8 proteins,9 and synthetic polymers,10 and (iii) in
combination with other materials as nanocomposites.11−13
Given the utility of these nanotubes, it is important to improve
the efficiency in the design of precursor amphiphiles and
assembly conditions by increasing the understanding of the
influence that each variable has on the self-assembled
morphology. To do this, we applied a rational design approach
for constructing novel chiral amphiphiles (Chart 1), and by
systematically varying the molecular parameters of the
amphiphiles, we were able to study the effect of structure-
Self-assembly of nanotubes through the helical twisting of
bilayer ribbons is driven largely by the molecular structure of
the amphiphile.14 Herein we report the synthesis and
characterization of a series of newly designed amphiphilic
compounds (R-Azo-R′-Asp, where R = 4, 7, or 10 and is the
number of alkyl units in the distal chain and R′ = 10 or 11 and
is the number of alkyl units in the proximal chain; Chart 1).
The profound effect of the headgroup and interfacial region on
the self-assembly of amphiphiles is known and is usually the
focus of efforts in the rational design of self-assembled
amphiphilic systems.1,15−17 Amino acids have often been used
as a headgroup or in a linking role as they provide multiple
functionality and high chiral fidelity in a cost-effective
manner.18−21 Despite limited use in the extant literature,22
single residue aspartic acid was selected for the headgroup, as
its polar chemistry provides multiple opportunities for
hydrogen bonding, while the amide linkage to the hydrophobic
Received: July 30, 2012
Revised: September 13, 2012
Published: September 14, 2012
© 2012 American Chemical Society
14172
dx.doi.org/10.1021/la3030606 | Langmuir 2012, 28, 14172−14179

Langmuir
Article
a
Chart 1. Molecular Structures of the Synthesized Amphiphiles
a
All compounds have an aspartic acid headgroup linked by an amide bond to the hydrophobic tail, which comprises an azo group between two alkyl
chains. The key compositional variables are the lengths of the alkyl chains, the proximal chain being 10 or 11 carbons long, and the distal chain being
4, 7, or 10 carbons long.
chain provides a strongly directional interaction between
neighboring molecules. Importantly, this directional interaction
is adjacent to the chiral center and within the interfacial region
between the hydrophilic head and hydrophobic tail of the
amphiphile.23,24 Such organization of the headgroup and its
connection to the bent, hydrophobic tail was designed to drive
the formation of helically based nanotubes through molecular-
chirality-directed packing within the bilayer ribbons. In this
mechanism neighboring molecules pack at a consistent nonzero
angle, with the direction of propagation of the packing angle
controlled by the molecular chirality, such chiral molecular
packing being expressed in the macroscopic helices that form
the tubes.25,26
Overall, the influence of the hydrophobic tail on the self-
assembly of amphiphilic molecules is less well understood than
the influence of the hydrophilic headgroup and frequently more
subtle. Azo groups are often used in self-assembled systems to
exploit their photoresponsive nature.27 In our molecular design
the azo group was incorporated within the hydrophobic tail to
provide a rigid kinked structure that provides the offset in
molecular packing that can be chirally controlled. Such strategic
molecular design supports both molecular-chirality-directed
packing and another potentially complementary helical tube
forming mechanism based on chiral symmetry breaking,
previously observed in well-researched diacetylenic amphi-
philes.28−30 Additionally, the azo group affords opportunities
for π−π stacking during self-assembly, thereby strengthening
the noncovalent interactions between hydrophobic tails in the
resulting aggregate. The azo linkages also provide the prospect
of using light absorbance spectroscopy to probe the
organization of molecules in the nanostructure.31,32
great importance in determining the overall aggregate structure.
This finding is in contrast to diacetylenic systems where the
length variations in proximal and distal chains were found to be
of more limited importance.33,34 This particular effect of the
hydrophobic tail is difficult to predict, especially when
considered as part of the entire complex amphiphile interaction,
and illustrates why such empirical research is still important in
the optimization of the design of amphiphilic molecules for self-
assembly into helically based nanotubes.
EXPERIMENTAL SECTION
■
Materials. All materials were used as received. O-(7-Azabenzo-
triazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate
(HATU) and 1-hydroxy-7-azabenzotriazole (HOAt) were purchased
from GL Biochem (Shanghai) Ltd. (China); AR grade methyl
cyclohexane, hexane, and thionyl chloride were obtained from Merck
Pty. Ltd. (Australia), and hydrochloric acid (37% v/v) was purchased
from Ajax Finechem Pty. Ltd. (Australia). All other reactants and
reagents were purchased from Sigma-Aldrich Pty. Ltd. (Australia) and
were AR grade where available. Deuterated solvents for NMR analysis
were manufactured by Cambridge Isotopes Laboratories, Inc. Other
solvents were AR grade obtained from Ajax Finechem Pty. Ltd.
(Australia). All water used for self-assembly was deionized using a
Barnstead E-pure water purification system operating at a resistance of
at least 18.0 MΩ.
Synthesis. Detailed synthetic procedures and molecular identi-
fication (1H NMR, 13C NMR, FTIR, and elemental analysis) for the
four amphiphiles 4-Azo-10-Asp, 7-Azo-10-Asp, 10-Azo-10-Asp, and 7-
Azo-11-Asp (Chart 1) and their precursors are in the Supporting
Information. An example of the synthetic procedure for 4-Azo-10-Asp
is presented here.
4-[4-(Butylphenyl)diazenyl]phenol. 4-Butylaniline (4.30 g, 28.8
mmol) and hydrochloric acid (1.0 M, 96 mL) were stirred for 1 h at
room temperature before cooling to less than 5 °C. A solution of
sodium nitrite (2.19 g, 32 mmol) in water (∼4 mL) was then added
dropwise. The resultant cold diazonium solution was then added
dropwise to a solution of phenol (3.0 g, 32 mmol) and sodium acetate
Given that the amphiphiles vary only in the alkyl chain
lengths either side of the azo group, we demonstrate how, for
this system, the length of the distal and proximal chains is of
14173
dx.doi.org/10.1021/la3030606 | Langmuir 2012, 28, 14172−14179

Langmuir
Article
(4.7 g, 58 mmol) in water (48 mL) that was also stirring at less than 5
°C. The mixture was stirred for a further 3 h at less than 5 °C before
neutralization with aqueous sodium hydroxide (2.0 M), precipitating
the crude product. The solid was then filtered and washed with
copious amounts of water before being allowed to dry thoroughly
under suction. It was then washed with methylcyclohexane. Final
purification occurred by dissolution in a minimum quantity of
chloroform followed by precipitation with methylcyclohexane. Orange
platelike crystals (mp 78.5 °C) were isolated in 73% yield.
Methyl 10-Bromodecanoate. 10-Bromodecanoic acid (2.46 g, 9.79
mmol) was dissolved in methanol (67 mL) containing p-
toluenesulfonic acid (1% w/v), and the mixture was left to stir
overnight at room temperature. The methanol was then removed by
evaporation and the residue dissolved in diethyl ether. This solution
was then neutralized with aqueous sodium bicarbonate solution (1%
w/v), and the organic fraction was washed three times with water,
before drying over sodium sulfate and subsequent filtration. The
solvent was then removed in vacuo to reveal a colorless oil (mp −8.3
°C), isolated in 92% yield.
10-{4-[4-(Butylphenyl)diazenyl]phenoxy}decanoate. A mixture of
4-[4-(butylphenyl)diazenyl]phenol (16.2 mmol), methyl 10-bromo-
decanoate (4.31 g, 16.2 mmol), potassium carbonate (4.49 g, 32.4
mmol), potassium iodide (0.166 g, 1 mmol), and acetone (80 mL) was
stirred at reflux under nitrogen for 1 day. The solution was cooled to
room temperature, and then water (80 mL) was added. The
precipitate was collected by filtration and washed with water. The
resulting crude product was purified by recrystallization from
acetonitrile producing a yellow-orange solid (mp 64.9 °C) that was
isolated in 86% yield.
10-{4-[4-(Butylphenyl)diazenyl]phenoxy}decanoic Acid. 10-{4-[4-
(Butylphenyl)diazenyl]phenoxy}decanoate (2.22 g, 5.1 mmol) was
stirred in tetrahydrofuran (9 mL) under nitrogen and heated until the
solid dissolved. To this solution was added potassium hydroxide (1.43
g, 26 mmol) dissolved in water (9 mL), and the mixture was refluxed
overnight. The product was extracted with a mixture of diethyl ether
and tetrahydrofuran (1:1 v/v) and acidified by washing with aqueous
hydrochloric acid (1.0 M). The organic phase was then washed once
with water and then once with brine before drying over magnesium
sulfate, filtration, and subsequent evaporation of the solvents. The
isolated crude product was purified by dissolution in the minimum
quantity of hot ethyl acetate and then precipitated with hexane. The
solid was filtered and washed with hexane, revealing orange crystals
(mp 113.7 °C), isolated in 91% yield.
washing process by adding tetrahydrofuran. The organic phase was
then dried over magnesium sulfate and filtered before the solvent was
removed by evaporation. Purification was by recrystallization from
acetonitrile, revealing an orange solid (mp 97.4 °C), isolated in 87%
yield.
10-{4-[(4-Butylphenyl)diazenyl]phenoxy}decanoylaspartic Acid
(4-Azo-10-Asp). 10-{4-[(4-Butylphenyl)diazenyl]phenoxy}-
decanoylaspartic acid dimethyl ester (0.622 g, 1.1 mmol) was
dissolved in tetrahydrofuran (2 mL) and stirred under nitrogen. To
this was added aqueous potassium hydroxide (5.5 M, 2 mL), and the
mixture was refluxed overnight. Ethyl acetate/tetrahydrofuran (1:1 v/
v, 100 mL) was added, and the mixture was acidified with hydrochloric
acid (1.0 M, 2 × 100 mL). The organic phase was then washed once
with water and then once with brine before drying over magnesium
sulfate, filtration, and then subsequent evaporation of the solvents. The
isolated product was dissolved in the minimum quantity of ethyl
acetate and then precipitated with hexane. The precipitate was then
filtered under vacuum, revealing an orange solid in 74% yield.
Self-Assembly. Heated Self-Assembly Procedure. The amphi-
phile was dissolved in methanol (2.5 mL, 0.8 mM) by sonication at 63
°C. To this was added heated pure water (7.5 mL; 18.0 MΩ; 63 °C),
and sonication continued at 65 °C while the solution slowly became
cloudy. After 1 h sonication was ceased, but heating continued for a
further 5 h. The dispersion was then slowly cooled to room
temperature.
Room Temperature Self-Assembly Procedure (RT). The amphi-
philes were dissolved (0.4 mM) in methanol (100 mL). Aliquots of
this solution (300 μL) were then diluted 10-fold with mixtures of
methanol and pure water (2700 μL; water = 18.0 MΩ) such that the
final solutions ranged in methanol concentration relative to water from
10% to 100% in increments of 10%. The solvent mixture for each
amphiphile having the largest blue-shift using UV−vis spectroscopy
was selected for subsequent imaging by TEM.
Instrumentation. Ultraviolet and Visible Light Spectroscopy.
Ultraviolet and visible light (UV−vis) spectroscopy was conducted on
methanolic aqueous dispersions of nanotubes, as prepared in the room
temperature self-assembly procedure. Spectra were recorded with a
Varian Cary 50 Scan UV−vis spectrophotometer using a quartz
cuvette with a 10 mm path length.
Circular Dichroism. The amphiphiles were self-assembled using the
heated procedure, and the dispersion was analyzed by circular
dichroism (CD) using a JASCO J-725 spectropolarimeter equipped
with a Peltier accessory to maintain a temperature of 25 °C. The
experiment was conducted using a SQ-grade cuvette, with a path
length of 10 mm, at a scanning rate of 100 nm/min, a bandwidth of 1
nm, and a response time of 1 s, using a single accumulation.
Transmission Electron Microscopy. Transmission electron micros-
copy (TEM) was conducted on methanolic aqueous dispersions, as
prepared in the self-assembly procedures. The dispersion was diluted
4-fold using the same solvent mixture used in self-assembly, and then 4
μL of the dilution was deposited onto Formvar-covered copper grids
and air-dried before imaging using a JEOL JEM-1200EX instrument
operated at 80 kV with a spot size of 3.
L-Aspartic Acid Dimethyl Ester Hydrochloride. Thionyl chloride
(30 mL, 150 mmol) was added dropwise to methanol (120 mL) which
was rapidly stirred at −10 °C . To this solution was added L-aspartic
acid (6.66 g, 50 mmol), and then the reaction mixture was allowed to
warm to room temperature before being left to stir overnight. The
solvent was removed in vacuo, and the residue was dissolved in
methanol and then reduced in vacuo on two further occasions to
remove residual thionyl chloride. Purification was via dissolution in a
minimum quantity of methanol followed by precipitation with diethyl
ether and then filtration. This purification procedure was then
repeated revealing a white solid in 95% yield.
10-{4-[(4-Butylphenyl)diazenyl]phenoxy}decanoylaspartic Acid
Dimethyl Ester. 10-{4-[4-(Butylphenyl)diazenyl]phenoxy}decanoic
acid (0.850 g, 2.0 mmol) was stirred under nitrogen in a minimum
quantity of ethyl acetate/tetrahydrofuran (1:1 v/v) to dissolve the acid
at room temperature. Once dissolved, N,N-disopropylethylamine
(DIEA) (0.85 mL, 5 mmol), 1-hydroxy-7-azabenzotriazole (HOAt)
(0.27 g, 2 mmol), and O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetrame-
thyluronium hexafluorophosphate (HATU) (950.5 mg, 2.5 mmol)
were added, and this mixture was stirred for a further hour. Then L-
aspartic acid dimethyl ester hydrochloride (0.79 g, 4 mmol) was
added, and the stirring under nitrogen continued overnight at room
temperature. The resultant solution was diluted in ethyl acetate/
tetrahydrofuran (1:1 v/v, 100 mL), washed with aqueous solutions of
sodium hydrogen sulfate (5% w/v, 2 × 50 mL) and sodium hydrogen
carbonate (5% w/v, 2 × 50 mL), and then finally washed with brine (2
× 50 mL). The volume of the organic phase was maintained during the
RESULTS AND DISCUSSION
■
The basic amphiphile design utilized aspartic acid headgroup
interactions and the geometry and intermolecular interactions
of the azo group within the hydrophobic tail to drive self-
assembly into nanotubes. The influence of these two
components on the self-assembled morphology was elucidated
by synthesizing amphiphiles that varied in distal (4, 7, and 10
carbons long) and proximal (10, and 11 carbons long) chain
lengths. The distal chain length was set by the choice of 4-
alkylaniline precursors, which were converted to aryldiazonium
salts before coupling to phenol to create the azo group. The
proximal chain was generated by the addition of R′-bromoalkyl
carboxylic acid (where R′ is 10 or 11) to the azo hydroxyl
group using Williamson ether synthesis, before the final
14174
dx.doi.org/10.1021/la3030606 | Langmuir 2012, 28, 14172−14179

Langmuir
Article
Table 1. Summary of Characteristics of Structures Self-Assembled from the Synthesized Amphiphiles
attachment of the aspartic acid headgroup using an amide
coupling to the carboxylic acid. The self-assembly of the
amphiphiles was conducted in aqueous methanolic solutions
using two methods, viz., heated method and room temperature
(RT) method. In the heated method each amphiphile (0.2 mM
in the final self-assembly mixture) dissolved in methanol was
self-assembled at 65 °C by the addition of water (MeOH/H2O,
25/75 v/v), and the structures were annealed through heat and
sonication. For the RT method of self-assembly the compounds
were dissolved in methanol at RT and precipitated by adding
water. In the final mixture, the amphiphile concentration was
0.04 mM and the amount of methanol relative to water varied
from 10% to 100% in increments of 10%. TEM, UV−vis, and
CD analyses were used to study the influence of the
compositional variables and the effect of temperature (65 °C
and RT) as well as the methanol concentration (10−100% v/v
for RT method) on the self-assembly.
ribbons also found. The nanotubes had an average outer
diameter (o.d.) of 81 11 nm and an average wall thickness of
28 6 nm, giving a calculated internal diameter (i.d.) of 25 nm.
The helical ribbons had an average ribbon width of 71 14 nm
and an average pitch of 472 72 nm. The helical basis of the
tubes was confirmed by many instances of hybrid structures
exhibiting both tubular and helical morphologies (Figure 1b).
Using the RT self-assembly method with a solvent mixture
containing 80% water, precipitation of 4-Azo-10-Asp produced
nanotubes (o.d. 87 8 nm, wall 32 3 nm, i.d. 23 nm) that
closely matched those self-assembled using the heated
procedure. However, flattened tubes with very large external
tube widths (587
85 nm) and very thin tube walls were
predominant in the RT self-assembly of 4-Azo-10-Asp, and
these were also assembled via helical ribbon intermediates
(width 671
129 nm). Figure 2 exhibits examples of all of
these structures including the denser, narrow diameter tubes
Transmission Electron Microscopy. The results of the
self-assembly for all four amphiphiles are summarized in Table
1 and in further detail in the Supporting Information (Table
S1). Using the heated procedure, the aggregates self-assembled
from 4-Azo-10-Asp were dominated by nanotubes (Figure 1a),
with some amorphous aggregate and right-handed helical
Figure 2. TEM image of tubes and helices self-assembled from 4-Azo-
10-Asp using the RT method and having 80% water in the solvent
mixture.
Figure 1. TEM images of helically based tubes self-assembled from 4-
Azo-10-Asp using the heated method.
14175
dx.doi.org/10.1021/la3030606 | Langmuir 2012, 28, 14172−14179

Langmuir
Article
(see the top left corner of Figure 2), a wide helical ribbon, and
wide flattened tubes. A couple of the flattened tubes also
showed the growth of new helical ribbon layers. These ribbons
had varying widths and consistent pitch angles, indicating that
new layers formed by ribbon widening rather than pitch
compression (the most prevalent process for these types of self-
assembled tubes1).
Both the narrow tubes and the flattened tubes self-assembled
from 4-Azo-10-Asp at RT exhibited a further organizational
level; they aggregated into star-shaped bundles (Figure 3). This
83 26 nm.) and amorphous material. Indeed, helical ribbons
became the preponderant self-assembled structure for the RT
method using 90% water in the solvent mixture. The changing
relative populations of self-assembled morphologies between
RT and heated self-assembly methods support that the helical
ribbons are a kinetic product, isolated in greater populations by
the rapid precipitation at RT. Meanwhile, nanotubes are the
thermodynamic product, found in higher population under
better-optimized annealing conditions at 65 °C. Despite a lack
of observed wide ribbons self-assembled from 7-Azo-10-Asp
using the RT procedure, the organized aggregates for 7-Azo-10-
Asp were also often bundled together in star-shaped
morphologies, another similarity to the structures of 4-Azo-
10-Asp.
Organized aggregates self-assembled from 10-Azo-10-Asp
were predominantly right-handed helical ribbons (Figure 5)
Figure 3. TEM images of bundles of narrow tubes (a) and wide,
flattened tubes (b) self-assembled from 4-Azo-10-Asp using the RT
method and having 80% water in the solvent mixture. The arrow in
(b) illustrates the possible transition from wide tubes to narrow tubes.
suggests they are related structures. Indeed, what appears to be
a conversion from a wide, flat tube to a thinner tube is seen in
the top left of Figure 3b. Previous research on the nanotubes
self-assembled from a diacetylenic lipid suggested that self-
assembly of the final helically based tube occurred from a
similar flattened tube intermediate.35 This transitional structure
was only found using a novel electron microscopy specimen
protocol35 and had not been found using standard techniques.
While observation of transitional structures is rare,35,36 the self-
assembly of helically based tubes is suggested to proceed
through intermediates that are not always seen due to the rapid
kinetics of the self-assembly.36 In the case of 4-Azo-10-Asp we
deduce, similarly to other related amphiphiles,19,37 the rapid
precipitation of self-assembled structures at RT enabled the
isolation of a higher energy intermediate, which was not found
when the sample was cured at elevated temperature and
thermodynamic equilibrium was approached.
Amphiphile 7-Azo-10-Asp self-assembled into similar mor-
phologies found for 4-Azo-10-Asp except that 7-Azo-10-Asp
produced significantly more right-handed helical ribbons
(heated method: width 75 14 nm, pitch 357 50 nm; RT
method using 90% water: width 67 23 nm, pitch 386 54
nm [Figure 4b]) and less nanotubes (heated method: o.d. 76
6 nm, wall 24 3 nm, i.d. 28 nm [Figure 4a]; RT method: o.d.
Figure 5. TEM image of helical ribbons self-assembled from 10-Azo-
10-Asp using the heated method.
and distorted nanotubes, but in much lower populations than
for 4-Azo-10-Asp and 7-Azo-10-Asp, and amorphous aggregates
were dominant. Shimomura et al.38 previously demonstrated a
link between the distal chain length and the aggregate
morphology for bilayer assemblies of azo amphiphiles, finding
a shift from fully interdigitated bilayers to partially interdigi-
tated bilayers when the distal chain became long enough to
interfere with headgroup organization.38 Therefore, it is
proposed that the distal chain for 10-Azo-10-Asp interferes
with headgroup packing, thus preventing the formation of fully
interdigitated bilayers and leading to its relatively disorganized
self-assembly.
Amphiphile 7-Azo-11-Asp provided contrast to the other
amphiphiles by increasing the proximal chain length by one
methylene unit. This was reflected in the self-assembled
structures, being dominated by high axial ratio twisted ribbons
using both the heated and RT methods (heated method: pitch
196 49 nm, Figure 6; RT method using 90% water: width 35
1 nm, pitch 148 38 nm) (note: the relatively high standard
deviation of the pitch is indicative of variation between ribbons
rather than large variations within a single ribbon). In addition,
7-Azo-11-Asp formed low populations of both helical ribbons
(heated method: width 81 7 nm, pitch 428 33 nm, Figure
6a; RT method: width 131 14 nm, pitch 607 12 nm) and
nanotubes, the nanotubes being of two distinct diameters using
Figure 4. TEM images of structures self-assembled from 7-Azo-10-
Asp, using the heated method (a) and using the RT method with 90%
water in the solvent mixture (b).
each method (heated method: 1, o.d. 68 9 nm, wall 24
2
nm; 2, o.d. 166 16 nm, wall 51 7 nm, Figure 6b; RT
14176
dx.doi.org/10.1021/la3030606 | Langmuir 2012, 28, 14172−14179

Langmuir
Article
Figure 6. TEM images of structures self-assembled from 7-Azo-11-Asp
using the heated method.
method: 1, o.d. 48 5.4 nm, wall 21 1.5 nm; 2, o.d. 92 7.6
nm, wall 31 2.1 nm).
Twisted ribbons and helical ribbons are closely related
structures that can transform from one to the other in a non-
first-order transition,39−41 the helical morphology being found
to have greater chiral order.42,43 The twisted ribbons self-
assembled from 7-Azo-11-Asp have a pitch roughly half that of
the helical ribbons from the same amphiphile, which provides
confirmation that these structures are related as full ribbon
rotations come at approximately the same length. (The twist
pitch is measured every half rotation of the ribbon, while the
helical pitch is measured at every full rotation.) The change
from the dominance of the helical aggregates for R-Azo-10-Asp
amphiphiles to twisted aggregates for 7-Azo-11-Asp suggests
that the difference in relative orientations of the headgroup and
the azo group for 7-Azo-11-Asp compared to the R-Azo-10-Asp
species33,44 has reduced the intensity of the chirality of the
interactions without necessarily decreasing the order of the
assemblies. Moreover, R-Azo-10-Asp amphiphiles self-as-
sembled into right-handed chiral aggregates, whereas the chiral
aggregates (twisted and helical ribbons) of 7-Azo-11-Asp were
left-handed. Selinger et al.45,46 showed that the handedness of
macroscopic shapes depends not only on the microscopic chiral
parameters, but also on the orientation of the molecular tilt to
the long edge of the ribbon. It is clear that a change in
alignment between the headgroup and the azo group induced
by the extra methylene unit in the proximal chain could alter
the orientation of the molecular tilt and explains the different
handedness of the aggregates for 7-Azo-11-Asp compared to R-
Azo-10-Asp. Further, small changes in tilt direction relative to
nearest neighbors can also alter the elastic properties of the
bilayer and consequently the diameter and pitch of the final
self-assembled nanostructure.45,46 This was used to elucidate
why multiple, distinct pitch angles for helical ribbons can be
found for a single amphiphile45,47 and is a reasonable
explanation for the two distinct tube diameters found for
amphiphile 7-Azo-11-Asp. The effect of the proximal chain
length on the self-assembled aggregates again illustrates the
importance of the interplay between the influences of the azo
group and the headgroup on self-assembly.
Figure 7. UV−vis spectra for 4-Azo-10-Asp monitoring the change in
spectral profile for aqueous methanolic solutions containing varying
percentages of methanol at RT (only 3 of 10 solvent ratios included
for clarity).
amphiphiles is the reduction in intensity of the n−π* transition
at 430 nm upon self-assembly. This band is formally forbidden
for the planar organization of the azo group but exists in
solution due to a twist in the group around the NN−C plane
induced by repulsive interactions between ortho hydrogens.49
As such, it can be deduced that the aggregation of the
chromophores in the organized self-assembled structures led to
a reduction in twist in the azo groups compared to those in
solution.
Of specific interest were the differences between the
spectroscopy results monitoring the self-assembly of 7-Azo-
10-Asp and those for 4-Azo-10-Asp. UV−vis results showed
that initial assembly occurred using 10% less water in the
solvent mixture, due to reduced solubility, and the blue-shift
was only 29 nm, compared to 37 nm for 4-Azo-10-Asp. The
reduced blue-shift is indicative of a differing interaction
between the aggregated 7-Azo-10-Asp chromophores compared
to those for 4-Azo-10-Asp.50 This was supported by circular
dichroism (CD) spectra for 7-Azo-10-Asp and 4-Azo-10-Asp
self-assembled using the heated procedure. Figure 8 shows an
opposite sign and a reduced intensity of CD absorbance for 7-
Azo-10-Asp compared to 4-Azo-10-Asp, respectively, indicative
of a reduced intensity of chiral azo interactions and opposing
chiral arrangements of the chromophore transition moments in
the aggregates.51 However, this variation in molecular packing
of the azo groups, caused by the difference in distal chain
lengths, is not reflected in the consistently right-handed helical
morphologies observed using TEM. Consequently molecular-
chirality-directed packing, driven by the chiral headgroup, sets
the aggregate chirality for both of these species, in spite of the
varying organization of the azo groups. Nonetheless, the effect
of the azo group is significant, and there is interplay between
the influence on the self-assembly driven by the interactions of
the headgroups and the azo groups. This is demonstrated by
the fact that the azo interactions for 7-Azo-10-Asp differ in
character from those for 4-Azo-10-Asp, reflected by the
spectroscopic data and the reduced number of helical ribbons
wound sufficiently tightly to form tubes.24 This suggests that
the influence on self-assembly of the azo group and the
UV−vis and CD Spectroscopy Characterization. UV−
vis was used to monitor the self-assembly of the amphiphiles
using the RT procedure. There was a blue-shift and
concomitant reduction of intensity of the main π−π* transition
upon precipitation of all amphiphiles (example for 4-Azo-10-
Asp in Figure 7, complete spectra of all amphiphiles in the
Supporting Information, Figures S17−S20), indicative of H-
aggregation of the chromophores.48 This side-by-side arrange-
ment of azo groups makes for a strong, rigid, organized,
noncovalent bond, providing a high degree of order to the
aggregate. Another important feature of the spectra of all four
14177
dx.doi.org/10.1021/la3030606 | Langmuir 2012, 28, 14172−14179

Langmuir
Article
ASSOCIATED CONTENT
■
S
* Supporting Information
Table summarizing self-assembly results; experimental proce-
dures and analyses; additional TEM images and UV−vis
spectra. This material is available free of charge via the Internet
at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail thomas.barclay@flinders.edu.au; Ph +61 8 8201 3823;
Fax +61 8 8201 2905.
Present Addresses
§Jiangsu Key Laboratory of Advanced Functional Polymer
Design and Application, Department of Polymer Science and
Engineering, College of Chemistry, Chemical Engineering and
Materials Science of Soochow (Suzhou) University, Suzhou
215123, P. R. China.
⊥Gelest Inc., 11 East Steel Road, Morrisville, PA 19067.
Figure 8. CD spectra of 4-Azo-10-Asp and 7-Azo-10-Asp self-
assembled using the heated method.
Notes
The authors declare no competing financial interest.
REFERENCES
headgroup was more complementary for 4-Azo-10-Asp than it
was for 7-Azo-10-Asp.
■
(1) Shimizu, T.; Masuda, M.; Minamikawa, H. Supramolecular
Nanotube Architectures Based on Amphiphilic Molecules. Chem. Rev.
2005, 105, 1401−1443.
(2) Fang, J. Ordered Arrays of Self-Assembled Lipid Tubules:
Fabrication and Applications. J. Mater. Chem. 2007, 17 (33), 3479−
3484.
(3) Spector, M. S.; Selinger, J. V.; Schnur, J. M. Chiral Molecular Self-
Assembly. In Materials-Chirality; Green, M. M., Nolte, R. J. M., Meijer,
E. W., Eds.; John Wiley & Sons, Inc.: Hoboken, NJ, 2004; pp 281−
372.
(4) Zhou, Y. Lipid Nanotubes: Formation, Templating Nanostruc-
tures and Drug Nanocarriers. Crit. Rev. Solid State Mater. Sci. 2008, 33
(3−4), 183−196.
(5) Price, R.; Patchan, M. Controlled Release from Cylindrical
Mircostructures. J. Microencapsulation 1991, 8 (3), 301−306.
(6) Chow, G. M.; Stockton, W. B.; Price, R.; Baral, S.; Ting, A. C.;
Ratna, B. L.; Schoen, P. E.; Schnur, J. M.; Bergeron, G. L.; Czarnaski,
M. A.; Hickman, J. J.; Kirkpatrick, D. A. Fabrication of Biologically
Based Microstructure Composites for Vacuum Field Emission. Mater.
Sci. Eng., A 1992, A158, 1−6.
(7) Yang, B.; Kamiya, S.; Shimizu, Y.; Koshizaki, N.; Shimizu, T.
Glycolipid Nanotube Hollow Cylinders as Substrates: Fabrication of
One-Dimensional Metallic-Organic Nanocomposites and Metal Nano-
wires. Chem. Mater. 2004, 16 (14), 2826−2831.
(8) Zhou, Y.; Ji, Q.; Masuda, M.; Kamiya, S.; Shimizu, T. Helical
Arrays of CdS Nanoparticles Tracing on a Functionalized Chiral
Template of Glycolipid Nanotubes. Chem. Mater. 2006, 18, 403−406.
(9) Wilson-Kubalek, E. M.; Brown, R. E.; Celia, H.; Milligan, R. A.
Lipid Nanotubes as Substrates for Helical Crystallization of Macro-
molecules. Proc. Natl. Acad. Sci. U. S. A. 1998, 95, 8040−8045.
(10) Jintoku, H.; Okazaki, Y.; Ono, S.; Takafuji, M.; Ihara, H.
Incorporation and Template Polymerization of Styrene in Single-
Walled Bilayer Membrane Nanotubes. Chem. Lett. 2011, 40 (6), 561−
563.
CONCLUSION
■
An investigation was made into the differences in the self-
assembly of four closely related amphiphiles with aspartic acid
headgroups and an azo group contained within the hydro-
phobic tails, varying only in proximal and distal chain lengths.
These amphiphiles were designed to self-assemble into
nanotubes using molecular-chirality-directed packing, driven
by the headgroup interactions and supported by the
intermolecular interactions and geometry of the hydrophobic
tail. 4-Azo-10-Asp and 7-Azo-10-Asp self-assembled most
efficiently into nanotubes, having distal chains short enough
to allow fully interdigitated assembly, and a proximal chain
length that supported greater chiral order in the self-assembled
structure.
The temperature of the self-assembly was another important
variable, with the heated method used to highlight the
thermodynamic products. The similarity in dimensions and
character of the helical ribbon (357−472 nm pitch; 71−81 nm
width) and nanotube (68−81 nm diameter; 24−28 nm wall)
structures found for 4-Azo-10-Asp, 7-Azo-10-Asp, and 7-Azo-
11-Asp using the heated method is indicative that the same
forces drive their self-assembly. It is subtle changes in molecular
packing driven by distal and proximal chain length that caused
the variations in the populations of certain morphologies. The
RT method of self-assembly allowed the identification of helical
ribbons as a kinetic precursor in the thermodynamic formation
of nanotubes from 4-Azo-10-Asp and 7-Azo-10-Asp and the
isolation of a fascinating transitional intermediate nanotube
with much larger dimensions for 4-Azo-10-Asp.
The variation in assembly between the four amphiphiles
makes it clear that there is interplay between the interactions of
the azo group in the hydrophobic tail and the hydrophilic
headgroup. These influences must be balanced to provide the
optimal assembly, and consequently, for azo-based amphiphiles,
the lengths of the proximal and distal chains provide a means
optimizing molecular structure for production of the desired
self-assembled morphologies.
(11) Ji, Q.; Kamiya, S.; Jung, J.-H.; Shimizu, T. Self-Assembly of
Glycolipids on Silica Nanotube Templates Yielding Hybrid Nanotubes
with Concentric Organic and Inorganic Layers. J. Mater. Chem. 2005,
15, 743−748.
(12) Archibald, D. D.; Mann, S. Template Mineralization of Self-
Assembled Anisotropic Lipid Microstructures. Nature 1993, 364, 430−
433.
(13) Zhou, Y.; Kogiso, M.; He, C.; Shimizu, Y.; Koshizaki, N.;
Shimizu, T. Fluorescent Nanotubes Consisting of CdS-Embedded
14178
dx.doi.org/10.1021/la3030606 | Langmuir 2012, 28, 14172−14179

Langmuir
Article
Bilayer Membranes of a Peptide Lipid. Adv. Mater. 2007, 19 (8),
1055−1058.
(36) Ziserman, L.; Lee, H.; Raghavan, S. R.; Mor, A.; Danino, D.
Unraveling the Mechanism of Nanotube Formation by Chiral Self-
Assembly of Amphiphiles. J. Am. Chem. Soc. 2011, 133, 2511−2517.
(37) Kogiso, M.; Aoyagi, M.; Asakawa, M.; Shimizu, T. Semisolid
Phase Synthesis of Metal-Complexed Organic Nanotubes. Chem. Lett.
2010, 39 (8), 822−823.
(38) Shimomura, M.; Aiba, S.; Tajima, N.; Inoue, N.; Okuyama, K.
“Crystal Engineering” Based on Two-Dimensional Molecular
Assemblies. Relation between Chemical Structure and Molecular
Orientation in Cast Bilayer Films. Langmuir 1995, 11 (3), 969−976.
(39) Brizard, A.; Aime, C.; Labrot, T.; Huc, I.; Berthier, D.; Artzner,
F.; Desbat, B.; Oda, R. Counterion, Temperature, and Time
Modulation of Nanometric Chiral Ribbons from Gemini-Tartrate
Amphiphiles. J. Am. Chem. Soc. 2007, 129 (12), 3754−3762.
(40) Ziserman, L.; Mor, A.; Harries, D.; Danino, D. Curvature
Instability in a Chiral Amphiphile Self-Assembly. Phys. Rev. Lett. 2011,
106, 238105−1−238105−4.
(41) Ghafouri, R.; Bruinsma, R. Helicoid to Spiral Ribbon Transition.
Phys. Rev. Lett. 2005, 94, 138101−1−138101−4.
(42) Jung, J.-H.; John, G.; Yoshida, K.; Shimizu, T. Self-Assembling
Structures of Long-Chain Phenyl Glucoside Influenced by the
Introduction of Double Bonds. J. Am. Chem. Soc. 2002, 124,
10674−10675.
(43) Spector, M. S.; Singh, A.; Messersmith, P. B.; Schnur, J. M.
Chiral Self-Assembly of Nanotubules and Ribbons from Phospholipid
Mixtures. Nano Lett. 2001, 1 (7), 375−378.
(44) Xu, G.; Okuyama, K.; Ozawa, M.; Shimomura, M. The Odd-
Even Effect on Bilayer Structures of CnAzoCmN+Br−. Mol. Cryst. Liq.
Cryst. 1993, 237, 207−215.
(45) Selinger, R. L. B.; Selinger, J. V.; Malanoski, A. P.; Schnur, J. M.
Shape Selection in Chiral Self-Assembly. Phys. Rev. Lett. 2004, 93 (15),
158103.
(14) Yui, H.; Minamikawa, H.; Danev, R.; Nagayama, K.; Kamiya, S.;
Shimizu, T. Growth Process and Molecular Packing of a Self-
Assembled Lipid Nanotube: Phase-Contrast Transmission Electron
Microscopy and XRD Analyses. Langmuir 2008, 24 (3), 709−713.
(15) Schnur, J. M. Lipid Tubules: A Paradigm for Molecularly
Engineered Structures. Science 1993, 262, 1669−1676.
(16) Goldstein, A. S.; Lukyanov, A. N.; Carlson, P. A.; Yager, P.;
Gelb, M. H. Formation of High-Axial-Ratio-Microstructures from
Natural and Synthetic Sphingolipids. Chem. Phys. Lipids 1997, 88, 21−
36.
(17) Fuhrhop, J.-H.; Helfrich, W. Fluid and Solid Fibers Made of
Lipid Molecular Bilayers. Chem. Rev. 1993, 93, 1565−1582.
(18) Boettcher, C.; Schade, B.; Fuhrhop, J. H. Comparative Cryo-
Electron Microscopy of Noncovalent N-Dodecanoyl- (D- and L-)
Serine Assemblies in Vitreous Toluene and Water. Langmuir 2001, 17
(3), 873−877.
(19) Kogiso, M.; Aoyagi, M.; Asakawa, M.; Shimizu, T. Highly
Efficient Production of Various Organic Nanotubes with Different
Surfaces and Their Application to an Adsorbent. Soft Matter 2010, 6
(18), 4528−4535.
(20) Shao, H.; Gao, M.; Kim, S. H.; Jaroniec, C. P.; Parquette, J. R.
Aqueous Self-Assembly of L-Lysine-Based Amphiphiles into 1D n-
Type Nanotubes. Chem.Eur. J. 2011, 17 (46), 12882−12885.
(21) Shimizu, T.; Hato, M. Self-Assembling Properties of Synthetic
Peptidic Lipids. Biochim. Biophys. Acta 1993, 1147, 50−58.
(22) Imae, T.; Takahashi, T.; Muramatsu, H. Formation of Fibrous
Molecular Assemblies by Amino Acid Surfactants in Water. J. Am.
Chem. Soc. 1992, 114, 3414−3419.
(23) Polidori, A.; Michel, N.; Fabiano, A. S.; Pucci, B. Exotic Aqueous
Behavior of Synthetic Lipids: Formation of Vesicular Nanotubes.
Chem. Phys. Lipids 2005, 136, 23−46.
(24) Thomas, B. N.; Lindeman, C. M.; Corcoran, R. C.; Cotant, C.
L.; Kirsch, J. E.; Persichini, P. J. Phosphonate Lipid Tubules. II. J. Am.
Chem. Soc. 2002, 124 (7), 1227−1233.
(46) Selinger, R. L. B.; Selinger, J. V.; Schnur, J. M. Visualizing Chiral
Self-Assembly. Chaos 2004, 14 (4), S3.
(47) Zastavker, Y. V.; Asherie, N.; Lomakin, A.; Pande, J.; Donovan,
J. M.; Schnur, J. M.; Benedek, G. B. Self-Assembly of Helical Ribbons.
Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 7883−7887.
(25) Helfrich, W.; Prost, J. Intrinsic Bending Force in Anisotropic
Membranes Made of Chiral Molecules. Phys. Rev. A 1988, 38 (6),
3065−3068.
̈
(48) Scheibe, G. Uber Die Veranderlichkeit Der Absorptionsspektren
̈
in Losungen Und Die Nebenvalenzen Als Ihre Ursache. Angew. Chem.
1937, 50 (11), 212−219.
̈
(26) Selinger, J. V.; MacKintosh, F. C.; Schnur, J. M. Theory of
Cylindrical Tubules and Helical Ribbons of Chiral Lipid Membranes.
Phys. Rev. E 1996, 53 (4), 3804−3818.
(49) Kurita, N.; Tanaka, S.; Itoh, S. Ab Initio Molecular Orbital and
Density Functional Studies on the Stable Structures and Vibrational
Properties of Trans- and Cis-Azobenzenes. J. Phys. Chem. A 2000, 104
(34), 8114−8120.
(50) Song, X.; Perlstein, J.; Whitten, D. G. Supramolecular
Aggregates of Azobenzene Phospholipids and Related Compounds
in Bilayer Assemblies and Other Microheterogeneous Media:
Structure, Properties, and Photoreactivity. J. Am. Chem. Soc. 1997,
119 (39), 9144−9159.
(51) Jin, W.; Fukushima, T.; Niki, M.; Kosaka, A.; Ishii, N.; Aida, T.
Self-Assembled Graphitic Nanotubes with One-Handed Helical Arrays
of a Chiral Amphiphilic Molecular Graphene. Proc. Natl. Acad. Sci. U.
S. A. 2005, 102 (31), 10801−10806.
(27) Yager, K. G.; Barrett, C. J. Novel Photo-Switching Using
Azobenzene Functional Materials. J. Photochem. Photobiol. A: Chem.
2006, 182 (3), 250−261.
(28) Barclay, T.; Constantopoulos, K.; Matisons, J. Self-Assembled
Lipid Nanotubes by Rational Design. J. Mater. Res. 2011, 26, 322−335.
(29) Seifert, U.; Shillcock, J.; Nelson, P. Role of Bilayer Tilt
Difference in Equilibrium Membrane Shapes. Phys. Rev. Lett. 1996, 77
(26), 5237−5240.
(30) Pakhomov, S.; Hammer, R. P.; Mishra, B. K.; Thomas, B. N.
Chiral Tubule Self-Assembly from an Achiral Diynoic Lipid. Proc. Natl.
Acad. Sci. U. S. A. 2003, 100 (6), 3040−3042.
(31) Narayan, G.; Kumar, N.; Paul, S.; Srinivas, O.; Jayaraman, N.;
Das, S. Aggregation and Photoresponsive Behavior of Azobenzene-
Oligomethylene-Glucopyranoside Bolaamphiphiles. J. Photochem.
Photobiol., A 2007, 189 (2−3), 405−413.
(32) Ringsdorf, H.; Schlarb, B.; Vensmer, J. Molecular Architecture
and Function of Polymeric Oriented Systems: Models for the Study of
Organization, Surface Recognition, and Dynamics of Biomembranes.
Angew. Chem., Int. Ed. 1988, 27 (1), 113−158.
(33) Spector, M. S.; Selinger, J. V.; Singh, A.; Rodriguez, J. M.; Price,
R. R.; Schnur, J. M. Controlling the Morphology of Chiral Lipid
Tubules. Langmuir 1998, 14, 3493−3500.
(34) Singh, A.; Wong, E. M.; Schnur, J. M. Toward the Rational
Control of Nanoscale Structures Using Chiral Self-Assembly:
Diacetylenic Phosphocholines. Langmuir 2003, 19, 1888−1898.
(35) Mishra, B. K.; Garrett, C. C.; Thomas, B. N. Phospholpid
Tubulets. J. Am. Chem. Soc. 2005, 127, 4254−4259.
14179
dx.doi.org/10.1021/la3030606 | Langmuir 2012, 28, 14172−14179