Carbohydrate-coated supramolecular structures: Transformation of nanofibers into spherical micelles triggered by guest encapsulation

Article abstract of DOI:10.1021/ja070173p

Triblock rigid-flexible dendritic block molecules consisting of a rigid aromatic segment as a stem segment, carbohydrate-branched dendrons as a flexible head, and a hydrophobic alkyl chain were synthesized and characterized. The carbohydrate conjugate mol

Full text of DOI:10.1021/ja070173p

Published on Web 03/24/2007
Carbohydrate-Coated Supramolecular Structures:
Transformation of Nanofibers into Spherical Micelles
Triggered by Guest Encapsulation
Ja-Hyoung Ryu, Eunji Lee, Yong-beom Lim, and Myongsoo Lee*
Contribution from the Center for Supramolecular Nano-Assembly and Department of Chemistry,
Yonsei UniVersity, Seoul 120-749, Korea
Received January 9, 2007; E-mail: mslee@yonsei.ac.kr
Abstract: Triblock rigid-flexible dendritic block molecules consisting of a rigid aromatic segment as a stem
segment, carbohydrate-branched dendrons as a flexible head, and a hydrophobic alkyl chain were
synthesized and characterized. The carbohydrate conjugate molecule based on a methyl group as a
hydrophobic tail, in the solid state, self-assembles into a 1D nanostructure, whereas the molecule based
on a longer hydrophobic tail self-assembles into 2D nanosheets, as confirmed by X-ray scatterings. In
aqueous solution, however, both molecules were observed to self-assemble into carbohydrate-coated
cylindrical aggregates with a uniform diameter, as confirmed by dynamic light scatterings and transmission
electron microscopic (TEM) investigations. Notably, these cylindrical objects reversibly transformed into
spherical objects on addition of guest molecules. Investigation of the interactions of the carbohydrate-
coated nanostructures with E. coli cells showed that both nano-objects could immobilize bacterial cells,
while the degrees of immobilization were significantly dependent on the shape of nanostructure. These
results demonstrated that the supramolecular materials that are responsive to external stimuli can provide
novel opportunities to control many biological activities.
Introduction
reported, the self-assembly that plays a key role in living systems
provides a powerful approach toward the fabrication of complex
Controlled self-assembly of incompatible molecular compo-
nents can lead to a variety of nanostructures including spherical
micelles, vesicles, nanofibers, toroids, and tubes.1 The construc-
tion of supramolecular architectures by the self-assembly of
designed molecules is thus a subject of great current interest
and a challenging topic of interdisciplinary research in chem-
istry, biology, and materials science.2 In particular, self-
organization of carbohydrate conjugate molecules can endow
nanostructures with biological functions as multivalent ligands.3,4
Although many different designs of multivalent ligands such
as glycoprotiens,5 linear polymers,6 and dendrimers7 have been
nanoarchitectures. Precise control of molecular arrangements
of self-assembling molecules at the supramolecular level is
essential to get well-defined nanoscopic architectures as well
as desired biological functions.8 Introducing a rigid segment
into a self-assembling system has been reported to enhance
aggregation stability.9 In addition to stability, another important
issue regarding the preparation of these self-assembling systems
is their capability to respond to external stimuli such as pH,
temperature, and interaction with guest molecules and to interact
with biological receptors.10-12 To obtain precisely controlled
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J. AM. CHEM. SOC. 2007, 129, 4808-4814
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Carbohydrate-Coated Supramolecular Structures
A R T I C L E S
Scheme 1. Synthesis of the Carbohydrate Conjugate Aromatic Amphiphiles
and well-defined aggregates that are able to respond to external
stimuli such as interaction with guest molecules, however, the
more elaborate design of corresponding building blocks is
required, because the information determining their specific
assembly should be encoded in their molecular architecture.
Accordingly, we synthesized carbohydrate conjugate aromatic
molecules that can endow aggregates with enhanced stability,
external stimuli responsive character, and biological functions.
We present herein the formation of carbohydrate-coated nanofi-
bers from the self-assembly of carbohydrate conjugate aromatic
amphiphiles in aqueous solution and reversible switching
between fibers and spheres triggered by addition of guest
molecules. Notably, the specific multivalent interactions of
carbohydrate residues in the nanostructures with cognate binding
proteins in the pili of E. coli resulted in the shape-dependent
motility inhibition.
The design of a dendritic flexible chain was focused on the
construction of a carbohydrate conjugate segment that is soluble
in water and can have selectivity in bacterial cells. Mannose
containing dendron (3) was synthesized by glycosylation of
peracetylated bromo-D-mannose with ethylene glycol and change
of deprotection group from the acetyl to benzyl group, which
is more stable in basic conditions, and the subsequent convergent
route of oligoether dendron using etherification chemistry. The
aromatic core (4) prepared according to the procedures described
previously13 and then the subsequent etherification with tosylated
mannose conjugate dendron yielded 5, showing sufficient
solubility for further couplings of rigid conjugated building
blocks in the reaction medium. 6 was prepared from the Suzuki
coupling reaction with 5 and 4-hydroxy phenyl boronic acid in
the presence of Pd(0) catalysis. The final carbohydrate conjugate
molecules were synthesized by etherification of 6 and alkyl
halide and subsequent deprotection of the benzyl protecting
groups with Pd charcoal under H2 atmosphere. The resulting
carbohydrate conjugate aromatic amphiphiles (7 and 8) were
characterized by 1H and 13C NMR spectroscopy, elemental
analysis, and MALDI-TOF mass spectroscopy and were shown
to be in full agreement with the structures presented. As
Results and Discussion
Synthesis. The triblock rigid aromatic-flexible dendritic block
molecules consisting of a hydrophobic alkyl chain, a rigid
aromatic segment, and flexible carbohydrate conjugate dendrons
were obtained in a multiple synthesis from commercially
available starting materials (Scheme 1).
1
confirmed by H NMR spectroscopy, the ratio of the aromatic
protons of the rod block to the alkyl protons is consistent with
the ratio calculated. As shown in Figure 1, the MALDI-TOF
mass spectra of the carbohydrate conjugate aromatic molecules
exhibit three signals that can be assigned as the molecular ions,
together with the Na+ and K+ labeled molecular ions. The mass
corresponding to a representative peak in the spectrum is
matched with the calculated molecular weight of each of the
carbohydrate conjugate aromatic molecules.
(9) (a) Yoo, Y.-S.; Choi, J.-H.; Song, J.-H.; Oh, N.-K.; Zin, W.-C.; Park, S.;
Chang, T.; Lee, M. J. Am. Chem. Soc. 2004, 126, 6294-6300. (b) Discher,
B. M.; Won, Y. Y.; Ege, D. S.; Lee, J. C. M.; Bates, F. S.; Discher, D. E.;
Hammer, D. A. Science 1999, 284, 1143-1146. (c) Kukula, H.; Schlaad,
H.; Antoniett, M.; Fo¨ster, S. J. Am. Chem. Soc. 2002, 124, 1658-1663.
(d) Gohy, J.-F.; Lohmeijer, B. G. G.; Varshney, S. K.; Decamps, B.; Leroy,
E.; Boileau, S.; Schubert, U. S. Macromolecules 2002, 35, 9748-9755.
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2646. (b) Guo, X.; Szoka, F. C. Acc. Chem. Res. 2003, 36, 335-341. (c)
Johnsson, M.; Wagenaar, A.; Engberts, J. B. F. N. J. Am. Chem. Soc. 2003,
125, 757-760. (d) Che´cot, F.; Lecommandoux, S.; Gnanou, Y.; Klok, H.-
A. Angew. Chem., Int. Ed. 2002, 41, 1339-1343. (e) Lee, M.; Lee, S.-J.;
Jiang, L.-H. J. Am. Chem. Soc. 2004, 126, 12724-12725.
Solid-State Structure. The self-assembling behavior of the
carbohydrate conjugate aromatic molecules in the bulk was
investigated by means of thermal optical polarized microscopy
(11) (a) Ryu, J.-H.; Kim, H.-J.; Huang, Z.; Lee, E.; Lee, M. Angew. Chem., Int.
Ed. 2006, 45, 5304-5307. (b) Ajayaghosh, A.; Chithra, P.; Varghese, R.
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Aggregation Behavior in Aqueous Solution. Dynamic light
scattering (DLS) experiments were performed with 8 in aqueous
solution to investigate the aggregation behavior.15 The CONTIN
analysis of the autocorrelation function showed a broad peak
corresponding to a hydrodynamic radius (RH) ranging from
several nanometers to hundreds of nanometers (Figure 3a). The
formation of cylindrical micelles was confirmed by the Kratky
plot that shows a linear angular dependence over the scattering
light intensity of the aggregates (Figure 3b).16 The evidence for
the formation of the cylindrical aggregates was also provided
by transmission electron microscopy (TEM) experiments (Figure
3c). The micrographs negatively stained with a 2 wt % aqueous
solution of uranyl acetate show discrete cylindrical objects with
a uniform diameter of about 9 nm and a length of several
hundred nanometers. Considering the extended molecular length
(5.4 nm), the diameter of 9 nm indicates that the docosyl chains
within the core are fully interdigitated with each other. On the
basis of these results, it can be considered that 8 self-assembles
into cylindrical micelles consisting of a hydrophobic core
surrounded by hydrophilic mannose units in contact with the
aqueous environment. Similar to 8, 7 also formed cylindrical
micelles with dimensions of several hundred nanometers long
and a diameter of 8 nm in aqueous solution, as evidenced by
DLS and TEM.17
Figure 1. MALDI-TOF mass spectra of (a) 7 and (b) 8.
and X-ray scatterings. The X-ray diffraction pattern of 7 based
on a methyl group as a hydrophobic tail displays three sharp
reflections with the ratio of 1:x3:2 in the low angle region that
correspond to a 2D hexagonal columnar structure with a lattice
constant of 4.2 nm (Figure 2a). Considering the lattice constant
and extended molecular length (2.7 nm by Corey-Pauling-
Koltun (CPK) molecular model), this dimension implies that
the rodlike rigid segments arrange axially with their preferred
direction within a cross-sectional slice of the column, in which
rigid aromatic building blocks pack in an interdigitated fashion
(Figure 2b).
In contrast, the X-ray diffraction pattern of 8 based on a longer
hydrophobic docosyl chain displays sharp reflections that
correspond to equidistant q-spacings and thus index to a lamellar
lattice. The layer thickness obtained from the X-ray diffraction
pattern appeared to be 8.3 nm, that is, much larger than the
estimated molecular length (5.4 nm by CPK model), indicating
that the hydrophobic parts are interdigitated (Figure 2b).14
The results described above demonstrate the capability of
manipulating the carbohydrate-coated supramolecular structure
by grafting flexible hydrophobic chains of different lengths to
the apex of the same rigid building block conjugated mannose
branched dendron. The variation in the supramolecular structure
can be rationalized by considering the microphase separation
between the dissimilar parts of the molecule and the space-
filling requirement of the flexible hydrophobic chains. Molecule
7 based on a methyl group at the end of the rod segment can be
packed with fully interdigitated fashion of the rod segments,
and the bulky dendritic geometry of the flexible segments is
likely to frustrate the 2D growth of a self-assembled structure,
which results in carbohydrate-coated 1D cylinders. Increasing
the length of the hydrophobic chain, however, prohibits the
interdigitaion of the rod segments due to phase separation
between aromatic rods and alkyl chains, resulting in the
formation of carbohydrate-coated 2D sheets.
Remarkably, these cylindrical objects transformed into spheri-
cal objects on addition of Nile Red above 1 mol % relative to
the carbohydrate conjugate molecules.18 As shown in Figure
4a, the relaxation time of a mixture solution of Nile Red and 8
in the correlation function is shorter than that of 8. The CONTIN
analysis of the correlation functions in DLS experiments with
the solution containing the guest molecule showed RH to be 7
nm (Figure 4b). These results indicate that the cylindrical
micelles change into spherical micelles on addition of the
hydrophobic guest molecule. The formation of spherical objects
was further confirmed by TEM (Figure 4c). The micrographs
revealed spherical objects with an average diameter of about
14 nm, which is in good agreement with the results obtained
by DLS. Upon removal of the guest molecule by extraction with
n-hexane, the original cylindrical objects were observed to be
fully recovered as confirmed by DLS and TEM, thus indicating
that this structural transformation is reversible depending on
the presence of hydrophobic aromatic substrates (Figure 4b).
This phenomenon can be rationalized by considering the
interruption of the packing of the rod segments by intercalation
of guest molecules. The Nile Red would intercalate between
the aromatic cores of the cylindrical micelles through hydro-
phobic and π-π interactions. This intercalation causes the
packing of the aromatic segments within the core to be loose.
As a result, the cylindrical micelles might break up into spherical
micelles (Figure 5). The intercalation of Nile Red in rod
segments of 8 was confirmed by using fluorescence spectros-
copy (Figure 4d). When the solution of 8 and Nile Red was
excited at 294 nm, where most of the radiation is absorbed by
8, the intensity corresponding to the emission of 8 significantly
(15) Gohy, J. F.; Lohmeijer, B. G. G.; Alexeev, A.; Wang, X. S.; Manners, I.;
Winnik, M. A.; Schubert, U. S. Chem.-Eur. J. 2004, 10, 4315-4323.
(16) Fujita, H. Polymer Solutions; Elsevier Science: New York, 1990.
(17) See the Supporting Information.
(18) The transformation behavior of 7 and 8 was shown to be essentially the
same. The transformation occurred by addition of a small amount of
hydrophobic guest molecules such as Nile Red, nitrobenzene, and 4-4′-
dinitrobiphenyl. See the Supporting Information.
(14) The layer structure of 8 was further confirmed by TEM. See the Supporting
Information.
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Figure 2. (a) X-ray diffraction patterns of 7 and 8 measured at 25 °C. (b) Schematic representation for the hexagonal columnar structure of 7 and the
lamellar structure of 8 in the solid state.
micelles seems to interfere with the compact rod packing and
break up the cylinders to form the spheres.
To investigate the amount of dye encapsulation, UV/vis
spectroscopic experiments were performed via addition of Nile
Red in an aqueous solution of carbohydrate conjugate aromatic
molecules. The absorbance at absorption maximum of Nile Red
was plotted against the molar ratio of dye and each of the
molecules 7 and 8. As shown in Figure 6, the absorbance
increases with an increase in the ratio up to a certain point at
which the absorbance does not change with further increment
of the ratio. Therefore, the maximum mol percent of dye loading
per each molecule in the aqueous phase can be estimated to be
approximately 5% and 30% for 7 and 8, respectively. These
results demonstrate that the micelles self-assembled by the
molecule 8 can encapsulate much more guest molecules than 7
because long hydrophobic tails can provide more hydrophobic
space for the guest molecules. The entrapment of a hydrophobic
guest within the micelles can be rationalized by the strong
hydrophobic and π-π associations between the guest and the
amphiphilic molecule, and the poor solubility of the guest
molecule in water.
Biological Activities as a Multivalent Ligand. Self-assembly
of the amphiphiles into supramolecular objects coated by
carbohydrates suggests that they may function as supramolecular
multivalent ligands. Toward this direction, the quantitative
precipitation assay with lectin concanavalin A (Con A) was
selected because Con A recognizes R-D-mannopyranoside, and
these systems have been extensively studied as a model of
multivalent interactions (Figure 7).20 Multivalent interactions
between mannose residues on the nanostructures and Con A
Figure 3. (a) The size distribution graph at scattering angle of 90° from
CONTIN analysis of the autocorrelation function of laser light scattering
that were fluorescently labeled with fluorescein resulted in the
mutual cross-linking, thereby inducing the precipitation of the
mixture.21 After centrifugation, the degree of precipitation could
be determined by measuring the fluorescence intensity of the
soluble Con A fraction. Both cylindrical and spherical micelles
showed a similar efficient precipitation capability, indicative
of strong binding of mannose-coated supramolecular structures
of aqueous solution of 8 (0.1 wt %). (b) Kratky plot (9) and linear fit (-).
(c) TEM image of 8 with negative staining.
decreases while exhibiting another emission at 620 nm corre-
sponding to Nile Red. This result indicates that energy transfer
takes place between the aromatic units of Nile Red and those
of 8 within the supramolecular micelles,19 suggesting that the
insertion of Nile Red between the rod parts of 8 in cylindrical
(20) (a) Osawa, T.; Matsumoto, I. Methods Enzymol. 1972, 28, 323-327. (b)
Woller, E. K.; Cloninger, M. J. Org. Lett. 2002, 4, 7-10.
(21) Strong, L. E.; Kiessling, L. L. J. Am. Chem. Soc. 1999, 121, 6193-6196.
(19) Ryu, J.-H.; Lee, M. J. Am. Chem. Soc. 2005, 127, 14170-14171.
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Figure 4. (a) Autocorrelation functions and (b) size distribution graph at scattering angle of 90° from CONTIN analysis of the autocorrelation function of
laser light scattering of aqueous solution of 8 (0.1 wt %), mixture solution (0.1 wt %) of 8 and Nile Red (30 mol % relative to 8), and solution after
extraction of Nile Red. (c) TEM image for the spherical micelles (8‚Nile Red) with negative staining. (d) Emission spectra of the aqueous solution of 8 and
of 8‚Nile Red aqueous solution (0.1 wt % Nile Red; 30 mol % relative to 8; excitation wavelength 294 nm; inset: magnification between 550 and 700 nm).
Figure 5. The schematic representation of the reversible transformation of cylindrical micelles into spherical micelles.
to Con A. It should be noted that the precipitation was reversible
when a high excess of methyl mannoside (1000×) was added
to the precipitates. In contrast, a galactose, nonspecific to Con
A, was not able to reverse the precipitation, indicating that the
interaction is specific to mannose units.
Interestingly, both cylindrical and spherical micelles appeared
to specifically bind to the multiple mannose binding proteins
(MBPs) of bacterial pili in Escherichia coli (ORN 178),
demonstrating that the mannose-coated objects are excellent
multivalent ligands toward the specific receptors on the cell
surface.22 As shown in Figure 8, a number of cylindrical and
spherical objects were clearly observed to be located along the
fibers, indicative of strong binding of the objects to the MBPs.
Notably, the shape and size of the objects were retained even
after binding to the bacterial pili, indicative of high stability of
the supramolecular objects. The strong binding between the
objects and the MBPs seems to be attributed to recognition of
multivalent R-D-mannopyranoside ligands on the surface of a
micellar object by the receptors located on the bacterial pili.
This binding event was observed to be specific to the R-D-
mannopyranoside-coated objects. To confirm the selective
binding to MBPs, ORN 208 E. coli cells lacking the MBPs were
incubated with the nano-objects. In contrast to the ORN 178
strain, no supramolecular objects appeared to be imaged through
TEM, indicating that the bacterial pili of the ORN 208 strain
are unable to mediate R-D-mannopyranoside selective binding.
These results demonstrate that carbohydrate-coated objects
designed as multivalent nanoscaffolds for use in selective
receptor binding can be constructed from self-assembly of
carbohydrate conjugate aromatic amphiphiles.
In addition, the spherical micelles (8‚Nile Red) can be used
as scaffolds for the selective labeling of a specific cell as the
micelles contain fluorescent dye. To investigate the potential
application as a cell labeling system, the spherical micelles (8‚
Nile Red) were added to the ORN 178 E. coli solution, and the
mixture was observed using a fluorescence microscope. As
(22) Harris, S. L.; Spears, P. A.; Havell, E. A.; Hamrick, T. S.; Horton, J. R.;
Orndorff, P. E. J. Bacteriol. 2001, 183, 4099-4102.
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Figure 8. TEM images with negative staining of a sectioned area of pili
of the E. coli ORN 178 strain bound with (b) cylindrical (8) and (c) spherical
micelles (8‚Nile Red). A portion of an E. coli is shown in the lower right.
Figure 6. (a) UV-vis spectrum for Nile Red dye encapsulated within 8
solution in water at various ratios of Nile Red/8 (mol/mol) (a range of ratio
from 0.05 to 1 mol/mol). (b) Absorbance intensity of Nile Red at 550 nm
as a function of the ratio of Nile Red/molecules (mol/mol).
Figure 7. (a) The quantitative precipitation assay with lectin concanavalin
A. Each value represents the mean ( s.d. (n ) 3).
shown in Figure 9a, ORN 178 E. coli could be easily detected
with red fluorescence of Nile Red, whereas no red fluorescence
was detected with cylindrical micelle (8) and when ORN 208
E. coli, which does not contain mannose-specific protein, was
treated with the dye containing spherical micelles. These results
imply that the carbohydrate-coated nanostructure with entrapped
dye molecules can have potential applications from target
selective nanocarriers, to labeling of specific protein on the cell
surface, to sensing of a range of pathogens.
To investigate the effect of the shape of nanostructure as
multivalent ligands, nanofibers (8) and spherical micelles (8‚
Nile Red) were added to the ORN 178 E. coli solution,
respectively. Upon the addition of carbohydrate-coated objects,
we found on the optical microscope that both cylindrical and
Figure 9. Fluorescence microscope image of the E. coli ORN 178 strain
bound with spherical micelles (8‚Nile Red). (b) Bacterial motility inhibition
assay. Each value represents the mean ( s.d. (n ) 3).
spherical micelles could inhibit motility of the E. coli ORN178
strain (Figure 9b).6b The motility inhibition occurred im-
mediately after addition of micellar solution into E. coli
solutions. However, the degree of E. coli motility inhibition with
the spherical micelles (8‚Nile Red) was observed to be
significantly lower than that with the nanofibers (8). It seems
that multivalent interactions between the mannoses on the
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nanostructures and the MBPs cause intra-bacterial pili aggrega-
tion and that aggregation might interfere with flagella motion
followed by the inhibition of bacterial motility. Again, the
inhibition was observed to be reversible by the addition of
methyl mannoside in high excess, but not by nonspecific
galactose. Other evidence of specificity is that all of the
nanostructures were not able to inhibit motility of a bacterial
strain that lacks the MBPs (ORN208).
The results from the experiments described above, Con A
precipitation and the inhibition of E. coli motility, imply that
there are size dependencies on the outcome of the interaction.
Con A tetramer has dimensions of 6.7 × 11.3 × 12.2 nm,23
whereas E. coli cells are more than 100× larger than Con A.
We explain the difference in the outcome of the interaction as
the Con A being unable to distinguish the size between the short
(spherical) and long (cylindrical) nanostructures, as the size of
spherical micelle from 8‚ Nile Red is already similar to that of
Con A tetramer. In contrast, as also can be evidenced from TEM
observation, a comparatively large E. coli cell should feel the
nanostructures differently. The spherical micelles are too short
to cross-link the pili, whereas long cylinders are able to cross-
link the pili. As a result, the degree of motility inhibition of E.
coli can be manipulated by controlling the shape of the
nanostructure.
aqueous solution was investigated. In the bulk state, the
carbohydrate conjugate molecule based on a short hydrophobic
tail was observed to self-assemble into a carbohydrate-coated
columnar structure, whereas the molecule based on a long
hydrophobic tail self-assembled into a carbohydrate-coated sheet
structure. In aqueous solution, both amphiphiles self-assemble
into well-defined discrete carbohydrate-coated nanofibers with
a uniform diameter. Interestingly, these nanofibers reversibly
transform into a spherical micellar structure on addition of guest
molecules. Both objects functioned as supramolecular multi-
valent ligands for lectin, Con A, and the receptors on E. coli.
In particular, the spherical objects were shown to be applicable
for labeling specific protein on cell surface due to entrapment
of fluorescent dye molecules. In addition, motility inhibition
experiments showed that the shape of supramolecular architec-
ture has a significant effect on the biological activity. These
results demonstrate that the ability of supramolecular cylinders
to be in response to external stimuli can provide novel
opportunities to control many biological activities.
Acknowledgment. This work was supported by the Creative
Research Initiative Program of the Korean Ministry of Science
and Technology and Pohang Accelerator Laboratory, Korea.
E.L. thanks the Seoul Science Fellowship Program.
Conclusions
Supporting Information Available: Experimental details.
This material is available free of charge via the Internet at
http://pubs.acs.org.
New carbohydrate conjugate aromatic amphiphiles were
synthesized, and their self-assembling behavior in both bulk and
(23) Bouckaert, J.; Poortmans, F.; Wyns, L.; Loris, R. J. Biol. Chem. 1996,
271, 16144-16150.
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