Hierarchical self-assembly of chiral complementary hydrogen-bond networks in water: Reconstitution of supramolecular membranes

Article abstract of DOI:10.1021/ja010035e

Spontaneous formation of complementary hydrogen-bond pairs and their hierarchical self-assembly (reconstitution) into chiral supramolecular membranes are achieved in water by mixing amphiphilic pairs of glutamate-derived melamine 6 and ammonium-derivatize

Full text of DOI:10.1021/ja010035e

6792
J. Am. Chem. Soc. 2001, 123, 6792-6800
Hierarchical Self-Assembly of Chiral Complementary Hydrogen-Bond
Networks in Water: Reconstitution of Supramolecular Membranes
Takayoshi Kawasaki,† Maki Tokuhiro, Nobuo Kimizuka,* and Toyoki Kunitake‡
Contribution from the Department of Applied Chemistry, Faculty of Engineering, Kyushu UniVersity,
Higashi-ku, Fukuoka 812-8581, Japan
ReceiVed January 3, 2001
Abstract: Spontaneous formation of complementary hydrogen-bond pairs and their hierarchical self-assembly
(reconstitution) into chiral supramolecular membranes are achieved in water by mixing amphiphilic pairs of
glutamate-derived melamine 6 and ammonium-derivatized azobenzene cyanuric acid 4. Electron microscopy
is used to observe formation of helical superstructures, which are distinct from the aggregate structures observed
for each of the single components in water. In addition, a spectral blue-shift and induced circular dichroism
(ICD) with exciton coupling are observed for the π-π* absorption of the azobenzene chromophores. These
observations are consistent with the reconstitution of the hydrogen-bond-mediated supramolecular membrane
6-4. Spectral titration experiments indicate the stoichiometric integration of the complementary subunits with
an association constant of 1.13 × 105 M-1. This value is considerably larger than those reported for the artificial
hydrogen-bonding complexes in aqueous media. The remarkable reconstitution efficiency is ascribed to the
hydrophobically driven self-organization of the amphiphilic, linear hydrogen-bond networks in water. Molecular
structure of the complementary subunits plays an important role in the complexation process since it is restricted
by the photoisomerized cis-azobenzene subunit. On the other hand, thermally regenerated trans-isomer 4
undergoes facile complexation with the counterpart 6. The present reconstitution of supramolecular membranes
provides the first example of complementary hydrogen-bond-directed formation of soluble, mesoscopic
supramolecular assemblies in water.
Introduction
mentary hydrogen-bond pairs are also formed in organic gels,5
liquid crystals,6 and in molecular cocrystals.7 Especially, the
pair of melamine and cyanuric acid (or barbituric acid) has been
popularly used to create a variety of supramolecular motifss
such as linear tapes, crinkled tapes, and circular structures.7e,f
These architectures are mostly governed by the steric interactions
among substituents introduced in the component molecules.
However, formation of hydrogen-bond-directed assemblies has
been largely carried out in the nonaqueous media, due to the
highly deteriorating action of water molecules against hydrogen
bonding.
Generally, enthalpic gain by the formation of hydrogen bonds
in water is canceled by the enthalpy to break hydrogen bonds
which have been formed between these molecules and water.8
Therefore, hydrogen bonding in the aqueous media requires
integration of the other noncovalent interactionsssuch as
hydrophobic interactions or aromatic stackingsto compensate
for the entropic disadvantage. Previous efforts made for the
Biological supramolecular assemblies as exemplified by
nucleic acid multiplexes, multimeric proteins, nucleic acid-
protein complexes, and viruses provide a variety of chiral
superstructures in the mesoscopic range (ca. 10-1000 nm in
scale).1 They spontaneously self-assemble in aqueous media by
ingeniously employing multiple noncovalent interactionsssuch
as electrostatic interactions, hydrogen bonding, dipole-dipole
interactions, and hydrophobic association. Inspired by these
biological self-assemblies, construction of supramolecular ag-
gregates has been an area of active research.2 Especially,
multiple hydrogen bonds between complementary molecular
components are popularly used as driving force for the formation
of dimers or oligomeric aggregates in organic media.3,4 Comple-
* Corresponding author. E-mail: kimitcm@mbox.nc.kyushu-u.ac.jp.
† Current address: Graduate School of Bioscience and Biotechnology,
Tokyo Institute of Technology, Nagatsuda 4259, Midori-ku, Yokohama,
226-8501, Japan.
‡ Current address: Frontier Research System, RIKEN, Wako, Saitama,
351-0198, Japan.
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10.1021/ja010035e CCC: $20.00 © 2001 American Chemical Society
Published on Web 06/21/2001

Reconstitution of Supramolecular Membranes
J. Am. Chem. Soc., Vol. 123, No. 28, 2001 6793
complementary hydrogen bonding in water took advantage of
hydrophobic microenvironments which were provided by aro-
matic surfaces9 or by the interior of aqueous micelles.10 Hy-
drophobic interactions also played a pivotal role in the self-
association of ureidotriazine derivatives in water.11 On the other
hand, recent studies at the air-water interface showed that the
complementary hydrogen bonding is drastically facilitated,
compared to that observed at the surface of aqueous micelles
or bilayers.12-14 However to date, in situ formation of comple-
mentary hydrogen-bond pairs in water and their hierarchical self-
assembly into the soluble, mesoscopic supermolecules have not
been realized, despite the omnipresent examples and their
significance in biology.
Scheme 1
We have previously reported that the suitably designed
amphiphilic hydrogen-bond networks form supramolecular
nano-assemblies in aqueous15 as well as in organic media.16 In
the aqueous system, preformed hydrogen-bond pairs of quar-
ternary ammonium-derivatized cyanuric acids (hydrophilic
subunits) and alkylated melamines (hydrophobic subunits) are
maintained in the bilayer structure (supramolecular mem-
branes).15 The hydrogen-bond networks present in the bilayer
are stabilized by their stacking, similar to the case of nucleic
acid base pairs in DNA. These findings prompted us to
investigate their reconstitution in water. We herein provide an
example that the complementary hydrogen-bond networks are
in situ-formed in water and undergo hierarchical self-assembly
into supramolecular membranes. The amphiphilicities, chemical
structures of the complementary pairs, and the aqueous environ-
ment exert decisive roles in the complexation process and in
the structural characteristics of reconstituted assemblies.
Scheme 2
“amphiphilic” hydrogen-bond pairs further self-assemble into
the higher supramolecular architectures. As the complementary
hydrogen-bond subunits, we have chosen the amphiphilic pair
of melamine and cyanuric acid.15 Azobenzene chromophore was
introduced in the cyanurate subunit (4, Scheme 1), to spectro-
photometrically monitor the hybridization process. We have
reported that the incorporation of an aromatic segment dramati-
cally enhances the stability of the preformed hydrogen-bond
networks in water.15 Azobenzene chromophores in the bilayer
assemblies display unique spectral characteristics depending on
their orientation,17 and the trans-to-cis photoisomerization of
the chromophore provides a unique opportunity to investigate
the role of molecular stereochemistry in the complexation
process. As the hydrophobic subunit, L- or D-glutamate-
derivatized melamine 2C14-L-(or D)-Glu-Mela (6) was em-
ployed (Scheme 2). It is reported that self-assembly of chiral
amphiphiles often leads to the formation of helical nanostruc-
tures,18 and such a morphology is only available through the
hierarchical self-assembly of the complementary subunits.
Electron Microscopy. Transmission electron micrographs of
the single component 4 (a), 6(D-form) (b) and the mixtures of
6(D-)-4 (1:1) (c), 6(D-)-4 (3:1) (d) in water are shown in Figure
1. The melamine derivative 6 was added from ethanol stock
solutions, and the ethanol content in the presence of 6 was
adjusted to 10 vol %. Aqueous dispersion of 4 alone displayed
globular aggregate structures with diameters of 30-50 nm
(Figure 1a). On the other hand, when the ethanolic solution of
6(D-) was injected in water, irregular ellipsoidal aggregates with
long axes of 40-130 nm were observed (Figure 1b). As the
solution was homogeneous at this concentration, it seems that
the melamine and glutamate moiety served as a hydrophilic
group to ensure the solubility.
Results and Discussion
Complementary Molecular Subunits for the Hybridization
in Water. To promote the formation of complementary hy-
drogen bonds in water, we propose to use the complementary
subunits that acquire amphiphilicity upon formation of the
hydrogen bonds. It is expected that such in situ-formed
(9) (a) Constant, J. F.; Fahy, J.; Lhomme, J. Anderson, J. E. Tetrahedron
Lett. 1987, 28, 1777. (b) Rotello, V. M.; Viani, E. A.; Deslongchamps, G.;
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Y.; Conn, M.; Rebek, J., Jr. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 1208.
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(b) Nowick, J. S.; Chen, J. S.; Noronha, G. J. Am. Chem. Soc. 1993, 115,
7636. (c) Nowick, J. S.; Cao, T.; Noronha, G. J. Am. Chem. Soc. 1994,
116, 3285.
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A. J. M.; Sijbesma, R. P.; Meijer, E. W. Nature 2000, 407, 167.
(12) (a) Kurihara, K.; Ohto, K.; Honda, Y.; Kunitake, T. J. Am. Chem.
Soc. 1991, 113, 5077. (b) Ikeura, Y.; Kurihara, K.; Kunitake, T. J. Am.
Chem. Soc. 1991, 113, 7343. (c) Sasaki, D. Y.; Kurihara, K.; Kunitake, T.
J. Am. Chem. Soc. 1992, 114, 10994. (d) Taguchi, K.; Ariga, K.; Kunitake,
T. Chem. Lett. 1995, 701. (e) Cha, X.; Ariga, K.; Kunitake, T. J. Am. Chem.
Soc. 1996, 118, 9545. (f) Koyano, H.; Bissel, P.; Yoshihara, K.; Ariga, K.;
Kunitake, T. Chem. Eur. J. 1997, 3, 1077 and references therein.
(13) (a) Kitano, H.; Ringsdorf, H. Bull. Chem. Soc. Jpn. 1985, 58, 2826.
(b) Ahlers, M.; Ringsdorf, H.; Rosemeyer, H.; Seela, F. Colloid Polym.
Sci. 1990, 268, 132. (c) Ahuja, R.; Caruso, P.-L.; Mo¨bius, D.; Paulus, W.;
Ringsdorf, H.; Wildburg, G. Angew. Chem., Int. Ed. Engl. 1993, 32, 1033.
(d) Bohanon, T. M.; Denzinger, S.; Fink, R.; Paulus, W.; Ringsdorf, H.;
Weck. M. Angew. Chem., Int. Ed. Engl. 1995, 34, 58. (e) Ebara, Y.; Itakura,
K.; Okahata, Y. Langmuir 1996, 12, 5166.
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Am. Chem. Soc. 1996, 118, 8524.
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1993, 115, 4387. (b) Kimizuka, N.; Kawasaki, T.; Kunitake. T. Chem. Lett.
1994, 33. (c) Kimizuka, N.; Kawasaki, T.; Kunitake. T. Chem. Lett. 1994,
1399. (d) Kimizuka, N.; Kawasaki, T.; Hirata, K.; Kunitake, T. J. Am. Chem.
Soc. 1998, 120, 4094.
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H.; Kunitake, T.; March, A.; Lehn, J.-M. J. Chem. Soc., Chem. Commnun.
1995, 2103.
Surprisingly, when 6(D-) and 4 were mixed in water at an
equimolar ratio, helical superstructures with thicknesses of 14-
(17) (a) Shimomura, M.; Ando, R.; Kunitake, T. Ber. Bunsen-Ges. Phys.
Chem. 1983, 87, 1134. (b) Shimomura, M.; S. Aiba.; Tajima, N.; Inoue,
N.; Okuyama, K. Langmuir 1995, 11, 969. (c) Song, X.; Perlstein, J.;
Whitten, D. G. J. Am. Chem. Soc. 1997, 119, 9144.
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Lett. 1984, 1709.

6794 J. Am. Chem. Soc., Vol. 123, No. 28, 2001
Kawasaki et al.
Figure 2. UV-vis spectra (a) and CD spectra (b) of aqueous 4 in the
absence and presence of 6 (L-). [6(L-)] ) [4] ) 5 × 10-5 M, ethanol;
20 vol %, 20 °C.
(b) of 4 in the absence and presence of equimolar 6(L-) in water
(concentration of ethanol, 20 vol %). The hydrogen-bond-
mediated supramolecular membrane is stable toward lysis up
to the concentration of 50 vol % ethanol, as will be discussed
later. The absorption peaks of azobenzene chromophore near
240 and 360 nm are associated with the π-π* transition moment
along the short and long axes, respectively.17a The long-axis
absorption band of 4 alone in water is observed at 360 nm
(Figure 2a), and this λmax is identical with that observed for the
molecularly dispersed azobenzene chromophores in alcoholic
solution, in micellar aggregates, or in liquid crystalline bilayers.17a
Upon heating the aqueous dispersion from 30 to 80 °C, the
absorption displayed a gradual intensity increase up to 35% of
the initial intensity (data not shown). The observed hypo-
chromism at lower temperature is indicative of a weak electronic
interaction among azobenzene chromophores, which is charac-
teristically observed for micellar aggregates.17a
Interestingly, upon the addition of equimolar 6(L-), the
absorption peak of 4 at 360 nm showed an immediate blue-
shift to 330 nm (Figure 2a). Such a blue-shifted absorption has
been observed for ordered azobenzene bilayers17 and is ascribed
to the strong exciton interaction among the regularly aligned
transition dipoles in the assembly.19 This is consistent with the
observation of developed helical superstructures in Figure 1c.
Furthermore, a sharp peak is observed at 219 nm, in addition
to the shoulder component of azobenzene short-axis transition
at 240 nm. The 219-nm band is red-shifted compared to the
absorption λmax of 6(D-) alone in ethanol (208 nm), and this
also originates from the exciton interaction among the melamine
units aligned in the linear complementary hydrogen-bond
networks.19,20a
Figure 1. Electron micrographs of aqueous dispersions. (a) 4, (b)
6(D-), (c) 6(D-):4 ) 1:1, (d) 6(D-):4 ) 3:1. Content of ethanol, (a) 0
vol %, (b)-(d) 10 vol %. Samples were stained by uranyl acetate.
28 nm, widths of 30-50 nm, and pitches of 180-430 nm were
observed (Figure 1c). These helical structures are stably
dispersed in water and are apparently distinct from those
observed for the individual subunits (Figure 1a, b). In addition,
they are observed in the wide ethanol concentrations (from 2.5
to 50 vol %). We have reported that such helical superstructures
are typically formed from the highly ordered, chiral bilayer
membranes.18 Therefore, nanohelices in Figure 1c most likely
are comprised of supramolecular membranes that are hierarchi-
cally self-assembled from the in situ-formed, amphiphilic
complementary hydrogen-bond networks 6-4. A similar helical
superstructure was obtained when 6(L-form) was employed in
place of the D-form. Interestingly, when both components were
mixed at a molar ratio of 6(D-):4 ) 3:1, globular structures
characteristic of the self-aggregates of 6(D-) were observed, in
addition to the helical structures (Figure 1d). These observations
suggest that the complexation of complementary subunits is
stoichiometric and excess subunit molecules are not incorporated
in the reconstituted assembly.
Circular dichroism of these aqueous solutions was investi-
gated to demonstrate the presence of excitonic interactions
(Figure 2b). Naturally, no CD peak was observed for the achiral
subunit 4 alone in water. On the other hand, upon the addition
of subunit 6(L-) to the aqueous solution of 4, two intense couples
(19) (a) Kasha, M. In Spectroscopy of the Excited State; Bartolo, B. D.,
Ed.; Plenum Press: New York, 1976; p 337. (b) Kasha, M. Rawls, H. R.;
El-Bayoumi, M.; Pure Appl. Chem. 1965, 11, 371.
(20) (a) Tokuhiro, M. Masters Thesis, Kyushu University, 1998. (b) ICD
component for the azobenzene-short axis band (λmax at 240 nm) is not
included in these peaks since it is located in the longer wavelength and the
magnitude of exciton interaction among azobenzene short-axis transition
dipoles are weak, as reported for chiral azobenzene bilayers.22c
UV-Vis Spectra and Induction of Circular Dichroism.
Figure 2 displays UV-vis absorption spectra (a) and CD spectra

Reconstitution of Supramolecular Membranes
J. Am. Chem. Soc., Vol. 123, No. 28, 2001 6795
Figure 3. Schematic illustration of the hybridization of amphiphilic complementary hydrogen-bond networks 6-4 in water and their hierarchical
self-assembly into supramolecular membranes. Chirality in the melamine subunit 6 is expressed as helical superstructures of the reconstituted
assembly.
of positive and negative Cotton effects appeared. A mirror-
symmetric CD spectrum was obtained when 6 (D-) was
employed in place of the L-form. The positive and negative
peaks at 216 nm ([θ] ) + 9.1 × 104 deg‚cm2‚dmol-1) and 228
nm ([θ] ) - 1.8 × 105 deg‚cm2‚dmol-1), and those at 315 nm
([θ] ) + 4.0 × 104 deg‚cm2‚dmol-1) and 346 nm ([θ] ) - 2.8
× 104 deg‚cm2‚dmol-1) are characteristic of the exciton
coupling,21 and they indicate the presence of strong excitonic
interaction among the π-π* transition dipoles of melamine and
azobenzene chromophores, respectively. According to a semi-
quantitative application of Kasha’s molecular exciton theory,19
the blue-shift of azobenzene absorption observed upon mixing
the subunits (Figure 2a) is ascribed to the parallel orientation
of the chromophores in the assembly.
It is noteworthy that the exciton coupling is induced in the
absorption band of achiral azobenzene subunit. Apparently,
structural information of the chiral melamine subunit is ef-
fectively transferred to the azobenzene subunit. Generally, a
magnitude of induced circular dichroism (ICD) is inversely
proportional to the third power of the distance between a chiral
molecule and an achiral chromophore.23 The appearance of such
an intense exciton-coupled ICD, then, must be ascribed to the
regularly fixed orientation of azobenzene group against the
glutamate moiety and to the resultant extensive delocalization
of photoexcitation energy over the oriented azobenzene chro-
mophores. Such regular supramolecular organization of two
subunits should be possible only via the formation of com-
plementary hydrogen bonds. The positive and negative Cot-
ton effects at 216 and 228 nm are ascribed to the exciton
interaction among the melamine subunits,20 and are clearly
distinguishable from the CD spectrum observed for the self-
aggregates of 6(D-)(data not shown, [θ]201 ) + 6.5 × 104
deg‚cm2‚dmol-1, [θ]216 ) - 1.2 × 105 deg‚cm2‚dmol-1, 18 h
after injection of the ethanol solution in water). These spectral
observations, together with the formation of helical superstruc-
tures, clearly indicate that the highly ordered supramolecular
membranes are reconstituted by complementary hydrogen
bonding in water.
observed slow increase in the ICD intensity is indicative of the
molecular ordering process in the reconstituted supramolecular
membrane.
It is interesting to compare these spectral characteristics with
those observed for the preformed supramolecular mem-
branes.15 In the previous studies, the complementary pairs were
prepared by mixing equimolar subunits in organic solvents. The
complementary hydrogen-bond pairs obtained after the re-
moval of solvents were successively dispersed in water by
ultrasonication.15 Aqueous dispersion of the preformed su-
pramolecular membrane 6(L-)-4 thus prepared possessed an
absorption λmax at 339 nm and ICD intensities of [θ]330 ) +
1.6 × 104 deg‚cm2‚dmol-1 and [θ]365 ) - 4.8 × 103
deg‚cm2‚dmol-1, respectively (20 °C). The observed absorp-
tion blue-shift and the ICD intensities are less eminent compared
to those of the in situ-formed assembly (Figure 2). The larger
ICD intensities observed for the reconstituted assembly must
reflect the improved molecular orientation. The less ordered
molecular alignment in the preformed membrane 6(L-)-4 is also
reflected in the lower population of helical structures observed
by electron microscopy.15c Ultrasonication of aqueous bilayers
is known to produce defects,24 and the in situ complexation
technique, which is devoid of such a deteriorating effect, thus
provides supramolecular membranes with the improved molec-
ular order.
The present reconstitution process is schematically shown in
Figure 3. By mixing the suitably designed complementary
subunits 4, 6 in water, amphiphilic networks of the linear
complementary hydrogen bonds 6-4 are spontaneously formed,
and they simultaneously self-integrate into the supramolecular
membranes. Water plays an essential role to direct the hydrogen
bonding and their hierarchical molecular organization. At first,
the self-aggregates of 6 collide and fuse with the globular self-
aggregates of 4, presumably due to hydrophobic interactions.
(21) Harada, N.; Nakanishi, K. Circular Dichroic Spectroscopy: Exciton
Coupling in Organic Stereochemistry; University Science Books: Mill
Valley, CA, 1983.
(22) (a) Kunitake, T.; Nakashima, N.; Shimomura, M.; Okahata, Y.;
Kano, K.; Ogawa, T. J. Am. Chem. Soc. 1980, 102, 6644. (b) Kunitake, T.;
Nakashima, N.; Morimitsu, K. Chem. Lett. 1980, 1347. (c) Nakashima, N.;
Morimitsu, K.; Kunitake, T. Bull. Chem. Soc. Jpn. 1984, 57, 3253.
(23) Craig, D. P.; Power, E. A.; Thirunamachandran, T. Chem. Phys.
Lett. 1974, 27, 149.
In contrast to the observed immediate blue-shift in the
azobenzene absorption upon mixing the complementary sub-
units, the intensity of the corresponding ICD peaks observed
just after mixing was about 65% of the maximum value (Figure
2b), the intensity of which was attained after ca. 1.5 h. The
(24) Szoka, F., Jr.; Papahadjopoulos, D. Annu. ReV. Biophys. Bioeng.
1980, 9, 467.

6796 J. Am. Chem. Soc., Vol. 123, No. 28, 2001
Kawasaki et al.
Figure 6. Dependence of [θ]total on the molar ratio of complementary
subunits. [6(L-)] ) 5 × 10-5 M, ethanol; 20 vol %, 20 °C.
is consistent with the observed segregation of excess subunits
from the reconstituted helical superstructures (Figure 1d).
In Figure 6, ICD intensity ([θ]
) |[θ]315| + |[θ]346|)
total
obtained for 6(L-)-4 is plotted against the molar ratio of
[4/6(L-)]. The ICD intensity also reached a constant value near
the composition of 4/6(L-) ) 1/1. The magnitude of association
constant K was determined according to the Benesi-Hildebrand
equation and by the least-squares fitting.
Figure 4. UV-vis spectra of aqueous 4 at varied molar ratio of
6(D-). 4: 6(L-) ) (a) 1:0, (b) 1:0.2, (c) 1:0.4, (d) 1:0.6, (e) 1:0.8, (f)
1:1. [4] ) 2.5 × 10-5 M, ethanol; 5 vol %, 20 °C.
1/∆[θ]obsd ) ([1/K]∆[θ]total)/Cazo + 1/∆[θ]total
(1)
where
Cazo ) [4] in mol‚L-1
(2)
Analysis of the data in Figure 6 indicates that 4 binds to 6 (L-)
with an association constant of 1.13 × 105 M-1. This value is
significantly larger than those reported for the hydrogen bonding
in aqueous media (binding of hexylthymine to acetylpenthyl-
adenine in SDS micelle; K ) 48 M-1 10c
or at the air-water
)
interface (barbituric acid to a surface monolayer of dialkylated
melamine12f; 2530 M-1, 2,4,6-triaminopyrimidine to a cyanurate
lipid monolayer13e; 3500 M-1). It is clear that formation of the
complementary hydrogen-bond-mediated bilayer in aqueous
media is much facilitated, compared to the previously reported
aqueous and surface monolayer systems.
Figure 5. Dependence of absorption maximum (9) and its intensity
(O) on the molar ratio of complementary subunits. [4] ) 2.5 × 10-5
M, ethanol; 5 vol %, 20 °C.
Subsequently, complementary hydrogen-bond networks are
formed in the mixed aggregates. The presence of bulk water
directs the supramolecular organization, in which the more
hydrophobic melamine subunits are organized in the interior
of the assembly and the ammonium-containing counterparts
constitute the outer surface of the assembly. This amphiphilic
supramolecular organization satisfies both the solubility and
the maintenance of the complementary hydrogen-bond net-
works in water. These hydrogen bonds are effectively shielded
from the bulk water by their packing in the bilayer. The ob-
served hydrophobically driven supramolecular organization is
reminiscent of the biological self-assemblies and the protein
folding.
Determination of the Stoichiometry and Association
Constant. To confirm the stoichiometry and to determine the
association constant of the complexation process, spectral
titration experiments were conducted for the pair of 6(D-) and
4. Figure 4 displays absorption spectra of 4 (2.5 × 10-6 M) in
the presence of 6(D-) at varied molar ratios. With increasing
the molar ratio of 6(D-), absorption λmax initially located at 357
nm for 4 alone shifted to 332 nm, and this spectral blue-shift
was accompanied by an increase in the absorption intensity. In
Figure 5, absorption λmax of 4 and its peak intensity are plotted
against the molar ratio [6(D-)/4]. Both the spectral blue-shift
and absorbance increase leveled off near the equimolar ratio,
indicating that the reconstitution occurs stoichiometrically. This
Effect of Ethanol Content on the Molecular Organization.
Generally, water-miscible organic solvents such as ethanol cause
lyses of aqueous bilayer membranes.24,25 Since melamine sub-
units are injected into water from ethanol solutions, the effect
of ethanol concentration on the reconstituted bilayer was in-
vestigated. Figure 7 displays UV-vis absorption (a) and CD
spectra (b) of 6(L-)-4 that has been reconstituted at varied
ethanol concentrations. Below the ethanol content of 20 vol %,
absorption λmax of azobenzene chromophore is located at 330
nm (parallel chromophore orientation), while the absorption at
360 nm starts to increase above the ethanol content of 40 vol
%. When 50 vol % of ethanol is present, spectral intensities at
330 and 360 nm become almost identical. By further increasing
the ethanol content to 70 vol %, complete shift of the absorption
peak to 357 nm was observed. In addition, a sharp absorption
peak at 220 nm, which is ascribed to the melamines organized
in the hydrogen-bond network, showed a decrease upon increas-
ing the ethanol content and completely disappeared at 70 vol
%. The absorption peaks observed at 70 vol % ethanol (357
nm for azobenzene, 206 nm for melamine) are almost identical
(25) (a) Dalton, A. D.; Miller, K. W. Biophys. J. 1993, 65, 1620. (b)
Regen, S. L.; Czech, B.; Singh, A. J. Am. Chem. Soc. 1980, 102, 6638. (c)
Roks, M. F. M.; Visser, H. G. J.; Zwikker, J. W.; Verkley, A. J.; Nolte, R.
J. J. Am. Chem. Soc. 1983, 105, 4507.

Reconstitution of Supramolecular Membranes
J. Am. Chem. Soc., Vol. 123, No. 28, 2001 6797
Chart 1
bilayer membranes such as 7, which are lysed in the presence
of 20-30 vol % ethanol26a (see Chart 1).
We have reported that stable bilayer membranes were formed
in binary aqueous-organic media when the amphiphile contains
aromatic chromophores such as N-methyl-p-nitroaniline in the
headgroup.26 The amphiphile 8 adopts highly ordered “tilt”
orientation in the presence of 40-60 vol % ethanol, and its
molecular orientational order, as estimated from the intensity
of CD spectra, is superior to that observed in pure water.
Stabilization of diacetylene-containing bilayers by ethanol has
been also reported by other researchers.27 The improved
molecular alignment in the presence of ethanol may be a feature
common to the bilayers with enhanced hydrophobicity and
crystallinity.26 Strong stacking of complementary hydrogen-bond
networks and of azobenzene chromophores in 6-4 might be
relaxed with the moderate ethanol concentration, which lowered
the dielectric constant of the solvent and allowed their ordering
in the assemblies.
Figure 7. UV-vis (a) and CD (b) spectra of aqueous 6(L-)-4 at varied
ethanol concentrations. [4] ) [6(L-)] ) 5 × 10-5 M, 20 °C.
Effect of trans-to-cis Photoisomerization of Azobenzene
Unit on the Hybridization. The trans-to-cis photoisomerization
of azobenzene chromophores in bilayers is influenced by the
physical state of the membranes, and it is suppressed when
bilayers are in rigid, gel state.28 Figure 9 shows spectral
absorption changes of the aqueous single component 4 upon
UV-irradiation and the succeeding time dependence in the dark.
Before photoirradiation (Figure 9a), the absorption peak is
observed at 360 nm as described previously. Upon irradiation,
the peak intensity at 360 nm decreases and new peaks appear
at 430 and 326 nm, which are ascribable to the n-π* and π-π*
transitions of the cis-isomer, respectively (Figure 9b). This
spectral change was complete within 10 min. The photoirradi-
ated spectrum (Figure 9b) possesses an absorption component
around at 360 nm, which is attributable to the 4(trans) species
that remains in the dispersion. The fraction of cis-isomer in the
photostationary state is ca. 85 mol %, as estimated by the
absorption decrease at 360 nm. When the photoirradiated dis-
persion of 4 was kept in the dark, a slow increase in absorbance
at 360 nm was observed, and 74% of the original intensity was
attained in 25 h at 20 °C. Thus, the subunit 4 in the aqueous
dispersion undergoes both the trans-to-cis photoisomerization
and cis-to-trans thermal isomerization. The observed facile
Figure 8. Dependence of ICD intensity [θ]total on the ethanol
concentration.
to those of the isolated chromophores, and these observations
indicate that the supramolecular membrane is lysed at this
ethanol concentration.
Interestingly, the magnitude of ICD spectra [θ] total () |[θ]315
|
+ |[θ]346|) in 6(L-)-4 (Figure 7b) showed a unique dependence
on the ethanol concentration. It increased with ethanol concen-
tration and reached a maximum at ca. 40 vol % (Figure 8).
Further increase in ethanol content results in a decrease in the
[θ] total value, and it almost disappeared at 70 vol %. Similarly,
CD intensity at 225 nm reached a maximum value at the ethanol
concentration of 40 vol % and is considerably weakened at 70
vol %. Consistent with these spectral observations, the developed
helical superstructure was not observed by electron microscopy
when the ethanol content exceeded 60 vol %.
(26) (a) Kimizuka, N.; Wakiyama, T.; Miyauchi, H.; Yoshimi, T.;
Tokuhiro, M.; Kunitake, T. J. Am. Chem. Soc. 1996, 118, 5808. (b)
Kimizuka, N.; Tokuhiro, M.; Miyauchi, H.; Wakiyama, T.; Kunitake, T.
Chem. Lett. 1997, 1049.
As described previously, the intensity of the exciton-coupled
CD reflects regularity of chiral molecular assembly, and
therefore the reconstituted bilayer 6(L-)-4 possesses the highest
molecular orientational order at the ethanol content of 40 vol
%. This tendency is in remarkable contrast with the conventional
(27) (a) Schunur, J. M.; Ratna, B. R.; Selinger, J. V.; Singh, A.; Jyothi,
G.; Easwaran, K. R. K. Science 1994, 264, 945. (b) Yamamoto, T.; Satoh,
N.; Onda, T.; Tsujii, K. Langmuir 1996, 12, 3143.
(28) (a) Shimomura, M.; Kunitake, T. J. Am. Chem. Soc. 1987, 109,
5175. (b) Kunitake, T.; Okahata, Y.; Nakashima, N.; Shimomura, M.; Kano,
K.; Ogawa, T. J. Am. Chem. Soc. 1980, 102, 6642.

6798 J. Am. Chem. Soc., Vol. 123, No. 28, 2001
Kawasaki et al.
Figure 11. Time dependence of absorption increase after photoirra-
diation. (9): aqueous single component 4, absorbance at 360 nm. (O):
aqueous dispersion of 6(L-)-4, absorbance at 330 nm. 20 °C.
Figure 9. Effect of photoirradiation on the UV-Vis spectra of aqueous
subunit 4 and succeeding spectral changes. (a) before photoirradiation,
(b) just after photoirradiation, t ) 0, (c) t ) 5 h, (d) t ) 10 h, (e) t )
15 h, (f) t ) 20 h, (g) t ) 25 h. [4] ) 5.2 × 10-5 M, ethanol; 16 vol
%, 20 °C.
to-trans thermal isomerization. The π-π* transition peak
observed at 324 nm (t ) 0) showed an increase in the intensity,
and this was accompanied by a small spectral red-shift to 332
nm. This absorption λmax is distinct form that of the single
component 4 (trans, λmax 360 nm), and is characteristic to the
reconstituted bilayer 6(L-)-4(trans).
In Figure 11, the time course of the absorption increase at
330 nm is plotted together with those observed for the
photoirradiated single component 4 (λmax at 360 nm, Figure 9).
In the latter case, the cis-to-trans isomerization of aqueous 4
proceeds almost linearly with time (O). Interestingly, the
absorbance increase at 330 nm (9) almost coincides with the
time dependence of the cis-to-trans isomerization of 4 alone.
Therefore, the cis-to-trans thermal transition of the component
4 is the rate-determining step in the reconstitution process, and
the reconstitution proceeds selectively with the trans-form. It
is interesting that the cis-form is not recognized as a comple-
mentary subunit. Apparently, the ability to form regular mo-
lecular alignment is required in the reconstitution process.
On the other hand, when photoillumination was conducted
for the reconstituted bilayer of 6(L-)-4, absorption intensity
decrease (λmax at 332 nm) observed after photoirradiation (5
min) was only ca. 18% of the initial intensity (data not shown).
This is accompanied by a slight absorption increase at 440 nm
(n-π* band of the cis-form), and the spectrum was unchanged
even after the prolonged irradiation of 20 min. Clearly, trans-
to-cis photoisomerization of azobenzene chromophore is re-
markably suppressed, once the subunit 4 is integrated in the
supramolecular membrane 6(L-)-4. By keeping the irradiated
dispersion in the dark for 24 h, 96% of the initial absorption
intensity at 332 nm recovered. This confirms that the cis-isomer
formed in the bilayer is reversibly complexed with 6(L-), after
the thermal cis-to-trans isomerization.
Figure 10. Effect of the addition of 6(L-) to the UV-vis spectra of
aqueous 4. (a) a) after photoirradiation (the same spectrum as Figure
9b). b) equimolar 6(L-) was added to the solution (t ) 0). (b) time
dependence after the addition of 6(L-). 6(L-) was added at t ) 0. [4] )
[6(L-)] ) 5 × 10-5 M, ethanol; 20 vol %, 20 °C.
These observations are schematically illustrated in Figure 12.
Upon the mixing of aqueous 4(trans) and 6, supramolecular
membrane 6-4 is facilely reconstituted in water (Figure 12a).
Photoillumination of azobenzene unit in the single component
4(trans) causes trans-to-cis isomerization (Figure 12b). The cis-
isomer formed undergoes thermal isomerization back to the
trans-form (Figure 12c). When the melamine subunit 6 is added
to 4(cis), no-hybridization occurs with the cis-isomer (Figure
12d). On the other hand, the thermally regenerated subunit
4(trans) undergoes immediate hybridization with 6, and the
supramolecular membrane 6-4 is formed (Figure 12g). The
suppressed trans-to-cis photoisomerization observed for the
reconstituted bilayer 6-4 clearly indicates that the supramo-
lecular membrane is in the crystalline order (Figure 12h). Similar
inhibition of trans-to-cis isomerization has been also observed
photoisomerization of 4 is consistent with the formation of fluid,
micellar self-aggregates.
Figure 10a shows absorption spectral change of photoirra-
diated 4 after the addition of equimolar subunit 6(L-). A rapid
decrease in the spectral component at 350-450 nm was
observed (t ) 0), which is accompanied by a slight absorption
increase at 324 nm. The rapid spectral decrease is ascribed to
the hybridization between the coexisting 4(trans, λmax at 360
nm) and 6(L-), as inferred from their efficient complexation in
water (Figure 2). This is also supported by the increase at 324
nm, which is close to the λmax of aqueous 6(L-)-4. In Figure
10b, time dependence of the absorption spectra was recorded.
The intensity of the n-π* absorption band (cis-isomer, at 444
nm) showed gradual decrease, indicating the progress of cis-

Reconstitution of Supramolecular Membranes
J. Am. Chem. Soc., Vol. 123, No. 28, 2001 6799
Figure 12. Schematic illustration of the complexation between subunits 6 and 4. The effect of photoisomerization is also shown. (a) Supramolecular
membrane is readily reconstituted in water from 4(trans) and 6. (b) Photoillumination of aqueous 4 causes trans-to-cis photoisomerization. (c) The
photoisomerized 4(cis) undergoes slow thermal isomerization to the trans form. (d, e) Addition of 6 to aqueous 4(cis) leads to no hybridization.
Only the trans-isomer coexisting in the solution is reconstituted into the supramolecular membrane 6-4. (f) Thermal isomerization of 4(cis) to
4(trans) in the aqueous mixture. (g) Facile complexation between regenerated 4(trans) and 6. On the other hand, trans-to-cis photoisomerization of
4 is suppressed in the reconstituted bilayer 6-4 (h).
for the ordered assemblies of azobenzene-bilayers,28 and also
saturated solution of aqueous NaCl. The organic layer was separated
for the long-chain derivatives of thioindigo29 and stilbene
and dried over Na2SO4, and evaporation of the solvent afforded
chromophores30 in the monolayer films.
tetradecyl D-glutamate as an oily product. To this oil, 2-chloro-4,6-
diamino-1,3,5-triazine (2.5 g, 17.2 mmol) was added, and the mixture
was heated at 90 °C for 18 h. After cooling to room temperature, the
crude product was extracted with chloroform and chromatographed on
Conclusions
It is established that hydrogen-bond-mediated bilayer mem-
branes are readily reconstituted in water by mixing the suitably
designed complementary subunits. Both the single subunits form
self-aggregates in water, and the hybridization process should
involve collision and fusion of these aggregates as an initial
step. Formation of complementary hydrogen-bond networks is
remarkably facilitated in these mixed aggregates, and aqueous
environment plays an indispensable role to direct the mode of
hydrogen bonding into the linear, amphiphilic structure. They
hierarchically self-assemble in water into the chiral supramo-
lecular membranes, and stability of the hydrogen bonds is effec-
tively secured by the integration. Participation of the aqueous
environment to direct mesoscopic supramolecular assemblies
has been a marked feature for the biological supramolecular
assemblies. The present reconstitution of supramolecular mem-
branes in aqueous media, together with the solvophobically
directed mesoscopic supermolecules in organic media,16 provides
a new generation of supramolecular chemistry.
silica gel (30:1 f 20:1 CHCl3/methanol). Recrystalization from ethanol
gave colorless powder (1.0 g, 9%). TLC (silica gel, CHCl3) Rf ) 0.03;
IR (KBr) ν (N-H) 3470, 3380, ν (C-H) 2920, 2850, ν (CdO) 1730,
ν (triazine) 815 cm-1; 1H NMR (CDCl3) δ 0.7-1.0 (t, 6H, CH3), 1.0-
1.6 (m, 48H, CH2), 2.35 (m, 2H, NCHCH2), 3.9-4.3 (m, 7H, NCHCO,
CH2COO, OCH2), 4.81 (s, 4H, NH2), 5.4-5.7 (d, 1H, NH). Anal. Calcd
for C36H68O4N6 0.25 H2O: C, 66.17; H, 10.57; N, 12.86%. Found: C,
66.23; H, 10.53; N, 12.56%. The L-isomer was synthesized by re-
fluxing 2-chloro-4,6-diamino-1,3,5-triazine and tetradecyl L-gluta-
mate hydrochloride in the presence of KHCO3. Anal. Calcd for
C36H68O4N6: C, 66.63; H, 10.56; N, 12.95%. Found: C, 66.63; H,
10.63; N, 12.89%.
4,4′-Bis[(ω-bromopenthyl)oxy]azobenzene (2). p-[(ω-Bromopenthyl)-
oxy]aniline hydrobromide (1) was prepared according to a previously
reported procedure.17a Ice-cooled aqueous NaNO2 (1.7 g, 24.5 mmol)
was cautiously poured into a stirred solution of p-[(ω-bromopenthyl)-
oxy]aniline hydrobromide (8.4 g, 24.8 mmol) dissolved in 200 mL of
water-acetone mixture (1/1 by volume) containing hydrochloric acid
(35%, 2.7 g), and the mixture was kept below 5 °C. Separately, CuSO4‚
5H2O (6.3 g, 25.4 mmol) was dissolved in water (50 mL), and aqueous
ammonia (28%) was added until the precipitate formed was dissolved.
To the resultant dark blue solution, aqueous hydroxylamine hydro-
chloride (1.7 g, 24.9 mmol in 20 mL) was added to give a colorless
mixture. As soon as the resultant colorless solution was poured into
the diazonium salt solution, black precipitate was formed. Acetone was
then added to the viscous reaction mixture, and stirring was continued
for 1.5 h. After removal of acetone from the reaction mixture under
reduced pressure, the crude product was extracted with chloroform.
Purification was conducted by column chromatography on silica gel
(eluent CHCl3, first elute was collected) to give 2.3 g (36%) of a reddish
yellow powder: mp 122 f 141 °C (arrow indicates the liquid crystalline
region); TLC (silica gel, CHCl3) Rf 0.2; IR(KBr) ν (C-H) 2930, 2860
Experimental Section
Materials. Glutamate derivatives of melamine and azobenzene-
containing isocyanuric acid were synthesized according to Schemes 1
and 2. The structures of the intermediates and the final products were
confirmed by thin-layer chromatography, IR and NMR spectroscopies,
and elemental analysis. Isocyanuric acid (Tokyo Kasei), CuSO4‚5H2O
(Kishida Chemical), 2-chloro-4,6-diamino-1,3,5-triazine (Aldrich) were
purchased and used as received. Ethanol used for spectral measurements
was of spectral grade (Kishida Chemical).
2C14-D-Glu-Mela (6). Tetradecyl D-glutamate (5) was synthesized
according to the reported procedure.31 The ester (5) (67 mmol) was
dissolved in chloroform and washed with aqueous Na2CO3 and with a
cm-1, ν (NdN) 1680 cm-1, ν (C-O-C) 1250 cm-1
.
(29) Whitten, D. G. J. Am. Chem. Soc. 1974, 96, 594.
(30) Whitten, D. G. Acc. Chem. Res. 1993, 26, 502.
(31) Asakuma, S.; Okada, H.; Kunitake, T. J. Am. Chem. Soc. 1991,
113, 1749.

6800 J. Am. Chem. Soc., Vol. 123, No. 28, 2001
Kawasaki et al.
N-{5-[p-{p′-(5-Bromopenthyloxy)phenylazo}phenyloxy]penthyl}-
cyanurate (3). (2) (2.3 g, 4.5 mmol) and isocyanuric acid (0.6 g, 4.6
mmol) were dissolved in 150 mL of DMF. 1,8-Diazbicyclo[5.4.0]undec-
7-ene (DBU; 0.7 g, 4.6 mmol) was added to this solution, and the
reaction mixture was heated at 60 °C for 14 h. After the reaction was
terminated by the addition of water, the precipitate formed was filtered
off, and the product was applied for column chromatography on silica
gel (CHCl3 f 29:1 CHCl3/methanol) to give yellow powder of (0.25
g, 10%): mp 210-215 °C; TLC(CHCl3/CH3OH, 29:1) Rf 0.71; IR-
(KBr) ν (N-H) 3200, 3050 cm-1, ν (C-H) 2930, 2860 cm-1, ν
Measurements. UV-vis spectra were measured on JASCO V-570
or Shimadzu UV-2200 spectrophotometer. Circular dichroism spectra
were obtained on JASCO J-720 spectropolarimeter. Transmission elec-
tron microscopy (TEM) was conducted on a Hitachi-H600 instrument
by the negative staining method. 500 µL of aqueous dispersions was
mixed with 500 µL of 2% aqueous uranyl acetate, and the mixture is
applied to carbon-coated grids without ultrasonication. Infrared spectra
were measured with a Shimadzu FTIR model DR-8000 (KBr pellet,
resolution: 2 cm-1) spectrometer. Water was purified by Milli-Q sys-
tem. In the photoisomerization experiment of azobenzene unit, 500-W
Hg lamp equipped with a Toshiba UV D35 filter was used as a light
source.
Reconstitution Procedure. Stock solution of 4 (1 mM) was prepared
by dissolving the sample in pure water or in ethanol/water mixture
(1/1 by vol) by ultrasonication. Melamine derivatives were dissolved
in ethanol (1 mM) and 100 µL of the solution were injected in 2.9 mL
of water or water-ethanol mixtures. Then, aqueous dispersion of 4
was added to the solution, and they were mixed by hand-shaking. In
every case, time dependence of absorption and circular dichroism
spectra after mixing was followed. Specimens for electron microscopy
were prepared for dispersions in a stationary state. For spectral titration
measurement, ethanolic solutions of melamine subunits were first
injected into water, and then aqueous 4 stock solution was added to
each solutions to give various melamine/cyanuric acid ratio, and the
mixture were kept overnight before the measurement.
(CdO) 1770, 1680 cm-1, ν (NdN) 1680 cm-1, ν (C-O-C) 1250 cm-1
;
1H NMR (3:1 CDCl3/DMSO-d6) δ 1.1-1.2 (m, 12H, CH2), 3.42 (t,
2H, CH2Br), 3.6-4.2 (t + t, 6H, OCH2, N-CH2), 6.7-8.0 (d + d,
8H, ph), 11.35 (s, 2H, NH).
N-{5-[p-{p′-(5-Trimethylammonio-penthyloxy)phenylazo}-
phenyloxy]penthyl}isocyanurate Bromide (4). (3) (0.25 g, 0.45 mmol)
was dissolved in 50 mL of anhydrous THF. Gaseous trimethylamine
(2.0 g, 33.9 mmol) was then dissolved in the solution, and the mixture
was stirred for 9 days at room temperature. A precipitate formed with
the progress of the reaction was collected and extracted with ethanol.
After removing the solvent in vacuo, recrystalization from water
1
afforded a yellow powder of 4 (0.05 g, 18%). H NMR (DMSO-d6/
D2O/CD3OD) δ 1.3-2.1 (m, 12H, CH2), 3.25 (m, 11H, N-CH3,
N-CH2), 7.0-8.0 (d + d, 8H, ph). Anal. Calcd for C28H39O5N6Br‚
1.5H2O: C, 52.10; H, 6.54; N, 13.00%. Found: C, 52.20; H, 6.64; N,
12.75%.
JA010035E