Controlled self-assembly of carbohydrate conjugate rod-coil amphiphiles for supramolecular multivalent ligands

Article abstract of DOI:10.1021/ja055999a

Carbohydrate conjugate rod-coil amphiphiles were synthesized and their self-assembling behavior in aqueous solution was investigated. These amphiphiles were observed to self-assemble into supramolecular structures that differ significantly depending on the molecular architecture. The rod-coil amphiphiles based on a short coil (1) self-assemble into a vesicular structure, while the amphiphiles with a long coil (2) show a spherical micellar structure. In contrast, 3, based on a twin-rod segment, was observed to aggregate into cylindrical micelles with twice the diameter of molecular length scale. As a means to determine the binding activity to protein receptors of these supramolecular objects, hemagglutination inhibition assay was performed. The experiments showed that the supramolecular architecture has a significant effect on the binding activity. In addition, incubation experiments with Escherichia coli showed that mannose-coated objects specifically bind to the bacterial pili of the ORN 178 strain. These results demonstrate that precise control of the nano-objects in shape and size by molecular design can provide control of the biological activities of the supramolecular materials.

Full text of DOI:10.1021/ja055999a

Published on Web 10/28/2005
Controlled Self-Assembly of Carbohydrate Conjugate
Rod-Coil Amphiphiles for Supramolecular Multivalent
Ligands
Byung-Sun Kim, Dong-Je Hong, Jinyoung Bae, and Myongsoo Lee*
Contribution from the Center for Supramolecular Nano-Assembly and Department of Chemistry,
Yonsei UniVersity, Seoul 120-749, Korea
Received August 31, 2005; E-mail: mslee@yonsei.ac.kr
Abstract: Carbohydrate conjugate rod-coil amphiphiles were synthesized and their self-assembling
behavior in aqueous solution was investigated. These amphiphiles were observed to self-assemble into
supramolecular structures that differ significantly depending on the molecular architecture. The rod-coil
amphiphiles based on a short coil (1) self-assemble into a vesicular structure, while the amphiphiles with
a long coil (2) show a spherical micellar structure. In contrast, 3, based on a twin-rod segment, was observed
to aggregate into cylindrical micelles with twice the diameter of molecular length scale. As a means to
determine the binding activity to protein receptors of these supramolecular objects, hemagglutination
inhibition assay was performed. The experiments showed that the supramolecular architecture has a
significant effect on the binding activity. In addition, incubation experiments with Escherichia coli showed
that mannose-coated objects specifically bind to the bacterial pili of the ORN 178 strain. These results
demonstrate that precise control of the nano-objects in shape and size by molecular design can provide
control of the biological activities of the supramolecular materials.
folding and the formation of biological membranes.6 Extensive
efforts thus have been directed toward bioactive supramolecular
Introduction
Novel approaches to the development of artificial multivalent
carbohydrate conjugate objects remain important area of research
due to their strong and specific interactions with the receptor
proteins.1 Many different designs of multivalent ligands have
been reported, with a number of scaffolds including glycopro-
teins,2 linear polymers,3 and dendrimers.4 Alternatively, self-
assembly of amphiphilic molecules containing carbohydrate
moieties is known to play a role in efficient multivalent ligands.5
The self-assembly of incompatible molecular components
leading to microphase separation comprises a powerful approach
toward the fabrication of complex nanoarchitectures and plays
an essential role in living systems, for example, in protein
systems for exploration of novel properties and functions that
are difficult without specific assembly of molecular compo-
nents.7 Block molecules that mimic lipid amphiphilicity have
been proved to be promising scaffolds for nanometer-sized
aggregates with well-defined size and shape.8 Introduction of 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 nanometer-scale
aggregates is their capability to interact with biological recep-
tors.10 To obtain precisely controlled and well-defined aggregates
with biological functions, however, the more elaborate design
of corresponding building blocks bearing bioactive moieties is
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A R T I C L E S
Kim et al.
light scattering (DLS) and transmission electron microscopy
(TEM) studies demonstrated that 1 self-assembles into a R-D-
mannopyranoside-coated vesicular structure with an average
diameter of approximately 40 nm.10 Compound 2, based on a
long oligo(ethylene oxide) chain, was also observed to assemble
into R-D-mannopyranoside-coated spherical aggregates, but they
are smaller in size. DLS measurements of 2 showed that RH is
approximately 12 nm with a narrow size distribution. TEM
micrographs showed spherical aggregates that are roughly 18-
22 nm in diameter and are thus in accord with the DLS results
(Figure 3b). These dimensions in diameter of the aggregates,
however, correspond to approximately twice the extended
molecular length (10 nm by CPK modeling), implying that the
aggregates of 2 are micellar in nature. The formation of very
small aggregates of higher curvature could be the result of a
more cone-shaped conformation of the long chain-based rod-
coil molecules compared to 1. It has been predicted that the
self-assembly process is governed by geometrical constraints
of individual molecules.13 As expected, 2a based on a â-D-
galactopyranoside moiety also formed essentially the same
spherical micellar objects as those of 2.
In contrast, compound 3, based on a twin-rod segment, was
observed to aggregate into cylindrical micelles with twice the
diameter of molecular length scale. Dynamic light scattering
(DLS) experiments were performed with 3 in aqueous solution
(2 × 10-4 g/mL) in order to investigate this unique aggregation
behavior (Figure 2). CONTIN analysis of the autocorrelation
function shows a broad peak corresponding to an average
hydrodynamic radius of approximately 70 nm. The angular
dependence of the apparent diffusion coefficient (Dapp) was
measured because the slope is related to the shape of the
diffusing species.14 The slope was observed to be 0.03,
consistent with the value predicted for cylindrical micelles. The
formation of cylindrical micelles was further confirmed by a
Kratky plot that shows a linear angular dependence over the
scattering light intensity of the aggregates.15 TEM micrographs
of 3 clearly show cylindrical aggregates with a uniform diameter
of about 20 nm and lengths up to several micrometers (Figure
3c). Considering the extended molecular length (11 nm by CPK
modeling), the image indicates that the diameter of the cylindri-
cal objects corresponds to approximately twice the extended
molecular length. All of these data indicate that 3 self-assembles
into a cylindrical micellar structure consisting of aromatic cores
surrounded by R-D-mannopyranoside-functionalized coil seg-
ments. The cylindrical structure of this micelle might be partly
explained by the more tapered shape of individual molecules,
compared to 2, but a second driving force might be strong π-π
interactions among aromatic segments down the long axis of
the cylinders.16
Figure 1. Molecular structure of 1-3 and schematic representation of
vesicles and spherical and cylinderical micelles.
required, since the information determining their specific
assembly should be encoded in their molecular architecture.
Accordingly, we synthesized rod-coil amphiphiles bearing
carbohydrates at one end of the coil segment that can endow
aggregates with enhanced stability and biological functions. In
this paper, we report significant size and structural changes of
the carbohydrate-coated aggregates, from vesicles to cylindrical
micelles to spherical micelles, with only small variations in
molecular architecture of the rod-coil amphiphiles 1-3 (Figure
1). These aggregates were observed to function as multivalent
ligands in the presence of natural receptors.
Results and Discussion
The amphiphilic rod-coil molecules consisting of tetra(p-
phenylene) or di[tetra(p-phenylene)] as a rod segment and R-D-
mannopyranoside-fuctionalized oligo(ethylene oxide)s as a coil
segment were obtained in a multiple synthesis from com-
mercially available starting materials.10,11 For comparison, the
homologous amphiphile of 2 bearing â-D-galactopyranoside (2a)
instead of R-D-mannopyranoside was also synthesized and
characterized. The resulting rod-coil amphiphiles were char-
acterized by 1H and 13C NMR spectroscopy, elemental analysis,
and matrix-assisted laser desorption ionization time-of-flight
(MALDI-TOF) mass spectroscopy and were shown to be in full
agreement with the structures presented.
Self-assembly of the rod-coil amphiphiles into supramo-
lecular objects coated by carbohydrates suggests that they may
(12) The critical micelle concentrations (cmc) of the molecules, determined by
plotting the absorption intensity of a hydrophobic dye, Nile Red, in an
aqueous phase against amphiphile concentration, were 2.3 × 10-6 M, 5.1
× 10-6 M, and 4.8 × 10-7 M for 1, 2, and 3, respectively.
(13) Israelachvili, J. N. Intermolecular and Surface Forces; Academic Press:
London, 1992.
(14) 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.
(15) Bockstaller, M.; Ko¨hler, W.; Wegner, G.; Vlassopoulos, D.; Fytas, G.
Macromolecules 2000, 33, 3951-3953.
(16) (a) Percec, V.; Cho, W.-D.; Ungar, G. J. Am. Chem. Soc. 2000, 122, 10273-
10281. (b) Hoeben, F. J. M.; Jonkheijm, P.; Meijer, E. W.; Schenning, A.
P. H. J. Chem. ReV. 2005, 105, 1491-1546.
All of the molecules in aqueous solution showed an aggrega-
tion behavior with a relatively narrow size distribution, indicat-
ing well-equilibrated structures (Figure 2).12 Previously, dynamic
(11) (a) Lee, M.; Jang, C.-J.; Ryu, J.-H. J. Am. Chem. Soc. 2004, 126, 8082-
8083. (b) Lee, M.; Park, M.-H.; Oh, N.-K.; Zin, W.-C.; Jung, H.-T.; Yoon,
D. K. Angew. Chem., Int. Ed. 2004, 43, 6466-6468.
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Carbohydrate Conjugate Rod−Coil Amphiphiles
A R T I C L E S
Figure 2. (a) Hydrodynamic radius distributions of 1-3; (b) autocorrelation function of 3; (c) angular dependence of the diffusion coefficient for 3; (d)
Kratky plot (9) and linear fit of 3 in aqueous solution with concentration of 2 × 10-4 g/mL.
be used as supramolecular multivalent ligands. Toward this
direction, hemagglutination inhibition assay with lectin con-
canavalin A (Con A) was selected because Con A recognizes
R-D-mannopyranoside and these systems have been extensively
studied as a model of multivalent interaction.17-19 The minimum
inhibitory concentration (MIC) of R-D-mannopyranoside unit
to inhibit Con A-promoted erythrocyte (rabbit) agglutination
was determined for each object. Relative potency was calculated
from the ratio of the minimum inhibitory concentrations of the
object-bound R-D-mannopyranoside and methyl mannose as a
standard. As shown in Figure 4, all of the objects showed high
inhibitory potency in the hemagglutination assay, indicating that
multivalent interactions between Con A and R-D-mannopyra-
noside units occur. The relative potencies of 1 and 3 showed
800-fold and 1000-fold increases, respectively, over methyl
mannose, while 2 showed an 1800-fold increase. These results
suggest that the spherical micellar objects with higher curvature
are more efficient inhibitors than the vesicular and cylindrical
objects. Although the origin of this difference in relative activity
depending on the supramolecular objects is not clear at present,
the high curvature of the smaller spherical object seems to play
an important role in enhanced inhibitory potency.20,21
Further information on the Con A-supramolecular object
binding events was provided by transmission electron micros-
copy (TEM) experiments performed at the concentration of the
hemagglutination assay (Figure 5). The micrographs of 1-Con
A and 2-Con A mixtures show spherical aggregates with
diameters of 200 and 180 nm, respectively, that are much larger
than those of Con A (6.5 nm) and the supramolecular objects
(40 and 20 nm for 1 and 2, respectively), demonstrating that
closely packed object-Con A clusters are formed in solution.
On the other hand, the micrograph of 3-Con A mixture shows
(17) Osawa, T.; Matsumoto, I. Methods Enzymol. 1972, 28, 323-327.
(18) Woller, E. K.; Cloninger, M. J. Org. Lett. 2002, 4, 7-10.
(19) Strong, L. E.; Kiessling, L. L. J. Am. Chem. Soc. 1999, 121, 6193-6196.
(20) Lee, R. T.; Lee, Y. C. Glycoconjugate J. 2000, 17, 543-551.
Figure 3. TEM images of (a) 1, (b) 2 without staining, and (c) 3 with
negative staining.
(21) Schlick, K. H.; Udelhoven, R. A.; Strohmeyer, G. C.; Cloninger, M. J.
Mol. Pharm. 2005, 2, 295-301.
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A R T I C L E S
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Figure 4. Relative activity of Con A-induced hemagglutination inhibition
based on the minimum inhibitory concentration (MIC) of 1-3. (Each value
represents at least three experiments; n.d. ) not determined.)
Figure 6. TEM images with negative staining of sectioned area of pili of
the E. coli ORN 178 strain bound with (a) 1 and (b) 2a. A portion of an E.
coli is shown in the lower right.
objects of 2a showed no nonspecific object-Con A association,
suggesting that the observed binding activity is specific to the
R-D-mannopyranoside-coated objects.
Interestingly, the micellar objects appeared to specifically bind
to FimH adhesin of bacterial type 1 pili in Escherichia coli,
demonstrating that the objects are excellent multivalent ligands
toward the specific receptors on the cell surface. Type 1 pili
are heteropolymeric mannose-binding proteinaceous fibers that
protrude from the surface of many gram-negative bacterial
cells.22 The bioactive binding ability of the objects was then
evaluated by interactions of the spherical micellar object of 2
with bacterial cells via TEM imaging. ORN 178 E. coli strain
was used in experiments to confirm the binding of the
supramolecular objects to FimH.23 After incubating of the
bacterial strain with the objects, the suspensions were centri-
fuged to pellet the cells that were washed further to remove
unbound objects. As shown in Figure 6a, a number of spherical
objects were clearly observed to be located along the fibers,
indicative of strong binding of the objects to FimH strain.
Notably, the shape and size of the objects was retained even
after binding to the bacterial pili, indicative of high stability of
the supramolecular objects. The strong binding between the
objects and FimH 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. In the control experiments, the
Figure 5. TEM images of aqueous solutions of (a) 1 and Con A, (b) 2 and
Con A, and (c) 3 and Con A at the hemagglutination inhibition concentration.
cylindrical aggregates with a uniform diameter of 40 nm that
is nearly double compared to that of the supramolecular object
of 3 (Figure 5c). This result suggests that each cylindrical object
is surrounded by lectin proteins through multivalent interactions.
The control experiments with â-D-galactopyranoside-coated
(22) (a) Jones, C. H.; Pinkner, J. S.; Roth, R.; Heuser, J.; Nicholes, A. V.;
Abraham, S. N.; Hultgren, S. J. Proc. Natl. Acad. Sci. U.S.A. 1995, 92,
2081-2085. (b) Choudhury, D.; Thompson, A.; Stojanoff, V.; Langermann,
S.; Pinker, J.; Hultgren, S. J.; Knight, S. D. Science 1999, 285, 1061-
1066.
(23) 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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A R T I C L E S
â-D-galactopyranoside-coated spherical objects of 2a were used
to replace R-D-mannopyranoside-coated ones under essentially
the same experimental conditions, but no apparent binding was
observed (Figure 6b). This suggests that the mannose units
located on the micellar surface were responsible for the
multivalent binding. To confirm the selective binding to FimH,
ORN 208 E. coli cells lacking the FimH protein were incubated
with the nanocapsules.23 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 mul-
tivalent nanoscaffolds for use in selective receptor binding can
be constructed from self-assembly of carbohydrate conjugate
rod-coil amphiphiles.
scale. Consequently, these systems clearly demonstrate the
ability to regulate the carbohydrate-coated supramolecular
architecture, from vesicular to spherical micellar to cylindrical
micellar structures by systematic variation in the length of coil
and the number of rods. More important, these objects func-
tioned as supramolecular multivalent ligands for lectin, Con A,
and the receptors on E. coli. Furthermore, hemagglutination
inhibition assay showed that the supramolecular architecture has
a significant effect on the binding activity. These results
demonstrate that the ability to control and systematically alter
the features of supramolecular materials with molecular design
can provide novel opportunity to investigate the widespread roles
of multivalent binding in biological systems.
Acknowledgment. We gratefully acknowledge the National
Creative Research Initiative Programs of the Korean Ministry
of Science and Technology for financial support of this work.
We thank Professor P. E. Orndorff for providing E. coli strains
and Professor H. S. Kim for hemagglutination assay experi-
ments.
Conclusion
The carbohydrate conjugate rod-coil amphiphiles were
synthesized and their self-assembling behavior in aqueous
solution was investigated. These amphiphiles were observed to
self-assemble into supramolecular structures that differ signifi-
cantly depending on the molecular architecture. The rod-coil
amphiphiles based on a short coil (1) self-assemble into a
vesicular structure, while the amphiphiles with a long coil (2)
show a spherical micellar structure. In contrast, compound 3,
based on a twin-rod segment, was observed to aggregate into
cylindrical micelles with twice the diameter of molecular length
Supporting Information Available: Synthetic procedures,
characterization, and experimental procedures for hemaggluti-
nation inhibition assays (HIA), dynamic light scattering (DLS),
and transmission electron microscopy (TEM). This material is
available free of charge via the Internet at http://pubs.acs.org.
JA055999A
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