Helical assembly of azobenzene-conjugated carbohydrate hydrogelators with specific affinity for lectins

Article abstract of DOI:10.1021/la300098q

Carbohydrate-mediated interactions are involved in various biological processes via specific molecular assembly and recognition. Such interactions are enhanced by multivalent effects of the sugar moieties, and thus supramolecular sugar-assembly, i.e., spontaneous association of glycoamphiphiles, is a promising approach to tailor glycocluster formation. In this study, novel sugar-decorated nanofibers were successfully prepared by self-assembly of low molecular weight hydrogelators composed of azobenzene and disaccharide lactones. Circular dichroism measurement of the as-prepared hydrogels indicated that the azobenzene amphiphile containing a lactose moiety possessed (R)-chirality, while the maltose-azobenzene conjugate exhibited (S)-chirality, even though the cellobiose-conjugated azobenzene existed in an achiral form. This suggests that the chiral orientation of the chromophoric azobenzene depended on both the glycosidic linkages and the steric arrangement of hydroxyl groups in the conjugated carbohydrates. Lectin-binding and cell adhesion assays revealed that the nonreducing ends of the conjugated sugar moieties were exposed on the surfaces of self-assembled nanofibrous hydrogels, allowing them to be effectively recognized by the corresponding lectins. In addition, photoisomerization of azobenzene under ultraviolet irradiation induced the sol-gel transitions of the hydrogels. These results demonstrate that the reversibly transformed fibrous glycohydrogels show potential for application as carbohydrate-decorated scaffolds for cell culture engineering.

Full text of DOI:10.1021/la300098q

Article
pubs.acs.org/Langmuir
Helical Assembly of Azobenzene-Conjugated Carbohydrate
Hydrogelators with Specific Affinity for Lectins
Yukiko Ogawa,† Chiharu Yoshiyama,† and Takuya Kitaoka*,†,‡
†Department of Agro-Environmental Sciences, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University,
Fukuoka 812-8581, Japan
‡Biotron Application Center, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan
ABSTRACT: Carbohydrate-mediated interactions are involved
in various biological processes via specific molecular assembly
and recognition. Such interactions are enhanced by multivalent
effects of the sugar moieties, and thus supramolecular sugar-
assembly, i.e., spontaneous association of glycoamphiphiles, is a
promising approach to tailor glycocluster formation. In this
study, novel sugar-decorated nanofibers were successfully
prepared by self-assembly of low molecular weight hydrogelators
composed of azobenzene and disaccharide lactones. Circular
dichroism measurement of the as-prepared hydrogels indicated that the azobenzene amphiphile containing a lactose moiety possessed
(R)-chirality, while the maltose−azobenzene conjugate exhibited (S)-chirality, even though the cellobiose-conjugated azobenzene
existed in an achiral form. This suggests that the chiral orientation of the chromophoric azobenzene depended on both the glycosidic
linkages and the steric arrangement of hydroxyl groups in the conjugated carbohydrates. Lectin-binding and cell adhesion assays
revealed that the nonreducing ends of the conjugated sugar moieties were exposed on the surfaces of self-assembled nanofibrous
hydrogels, allowing them to be effectively recognized by the corresponding lectins. In addition, photoisomerization of azobenzene
under ultraviolet irradiation induced the sol−gel transitions of the hydrogels. These results demonstrate that the reversibly
transformed fibrous glycohydrogels show potential for application as carbohydrate-decorated scaffolds for cell culture engineering.
surfaces.15−17 Herein, we are interested in the self-assembly of
low molecular weight glycoconjugates into nanostructures to
design multivalent sugar clusters.
INTRODUCTION
Carbohydrates play essential roles in all living organisms, and
interactions between carbohydrates and cellular proteins govern
■
Carbohydrate-based hydrogelators can form porous, three-
a wide variety of life processes including bacterial and viral
dimensional entangled networks in water by hierarchical self-
attachment, immune systems, transplant rejection, fertilization,
assembly.18−25 Supramolecular hydrogelators are generally
amphiphilic low molecular weight compounds and have received
attention because of their unique self-assembly features. Such
small organic molecules, even at low concentration (less than ca.
0.05 wt %), spontaneously form various nanostructures, e.g.,
nanofibers, nanosheets, nanoribbons, helices, and bundles, that
can trap water molecules in interstitial spaces.18−39 The driving
forces for hierarchical self-assembly include intermolecular
interactions such as π−π stacking, hydrogen bonding, van der
Waals interactions, hydrophobic association, and electrostatic
attraction. In consequence, supramolecular glyco-based amphi-
philes that form self-assembled nanostructures are expected to
have the following advantages: (1) spontaneous hydrophobic
association is possible in aqueous environments, and sugar
moieties are exposed on the surfaces of the assemblies as
hydrophilic head groups; (2) supramolecular aggregations form
via physicochemical interactions, and thus a reversible sol−gel
transition is expected in response to external stimuli; (3)
and cell proliferation/differentiation.1,2 Despite such significant
functions, monovalent carbohydrates generally have a low
affinity for most proteins, with dissociation constants ranging
from 10−4 to 10−3 M. Nevertheless, carbohydrate−protein
interactions result in strong binding power and the specificity
required to mediate biological processes through multivalent
contact, i.e., the glycocluster effect.3−5
In all living systems, glycolipid conjugates form sugar
assemblies on cell surfaces via morphological changes such as
rafts and caveolae, and glycoproteins also possess sugar-clustered
architectures.6 Based on biomimetic approaches, many studies
have been carried out to develop sugar-planted arrays, aiming at
using cluster formation to overcome weak monovalent sugar−
protein binding. Glycoconjugated polymers,7−9 glycoden-
drimers,10 glycoliposomes,11,12 and sugar microarrays using self-
assembled monolayers (SAM)13,14 have been reported. Conven-
tional approaches used to prepare glycoconjugated materials
usually involve the formation of covalent bonds between sugar
residues and designed substrates. Recently, amphiphilic glyco-
lipids found in vivo have attracted attention because of their
spontaneous association to form micelles, bilayers, and vesicles
where dense populations of glyco moieties are present on their
Received: January 7, 2012
Revised: January 28, 2012
Published: February 17, 2012
© 2012 American Chemical Society
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Synthesis of Glycine−Azobenzene Conjugate. 2-Amino-N-p-
phenylazophenyl acetamide (Gly-Azo): To Boc-Gly (0.37 g, 2.1
mmol) in chloroform (10 mL) cooled to 0 °C was added 1-ethyl-3-
(N,N′-dimethylaminopropyl)carbodiimide hydrochloride (0.40 g;
2.1 mmol) and 1-hydroxbenzotriazole (0.30 g, 2.2 mmol), followed
by 4-aminoazobenzene (0.40 g, 2.0 mmol) in chloroform (20 mL).
The mixture was stirred for 2 h in an ice bath. The reaction medium
was allowed to gradually reach ambient temperature overnight. The
mixture was concentrated; the residue was redissolved in the minimum
amount of chloroform and then washed sequentially with deionized
water, 0.1 mol L−1 aqueous hydrochloric acid, deionized water, and
finally saturated aqueous sodium hydrogen carbonate. The organic
phase was dried over anhydrous sodium sulfate, filtered, and
concentrated. The concentrate was distilled under reduced pressure to
afford Boc-Gly-Azo. Finally, the Boc protecting group was removed
by treatment in trifluoroacetic acid/dichloromethane (1:1 by volume)
for 1 h. The resulting trifluoroacetic salt was neutralized with
triethylamine/chloroform and evaporated to dryness in vacuo to give
Gly-Azo as a yellowish solid. Yield: 0.457 g (85.6%). 1H NMR (CDCl3):
9.65 (s, 1H), 7.96−7.80 (m, 7H), 7.51−7.45 (m, 3H), 3.52 (s, 2H). 13C
NMR (CDCl3): 170.9, 152.7, 140.2, 130.7, 129.1, 124.1, 122.7, 119.4,
45.2. MALDI-TOF-MS (m/z): [M + H] = 255.12 (calcd), 255.58
(found); [M + Na] = 277.11 (calcd), 277.56 (found).
Synthesis of Disaccharide Lactone Derivatives. N-p-Phenyl-
azophenyl acetamide-[O-β-D-galactopyranosyl-(1→4)]-D-gluconamide
(LL-Gly-Azo): lactonolactone (LL) was prepared from lactose according
to a reported protocol.8,44 As-prepared LL (0.68 g, 2.0 mmol) and
Gly-Azo (0.51 g, 2.0 mmol) were dissolved in methanol and heated
under reflux for 5 h and then allowed to stand at room temperature to
yield an orange-yellow crystal precipitate. The precipitate was filtered
and then washed with methanol and chloroform to remove unreacted
reagents. The purified product was dried under vacuum to give 0.98 g
(82.7%) of yellowish crystals. The purity of the sample was confirmed
by TLC (silica gel F254 precoated plates). Tm: 217 °C with decomposi-
tion. 1H NMR (DMSO-d6): 10.00 (s, 1H), 8.11 (t, 1H), 7.79−7.91 (m,
6H), 7.53−7.60 (m, 3H), 5.47 (d, 1H), 5.32 (d, 1H), 4.96 (d, 1H), 4.85
(d, 1H), 4.61 (t, 1H), 4.35−4.41 (dd, 2H), 3.97 (d, 2H). 13C NMR
(DMSO-d6): 172.8, 168.0, 151.9, 147.5, 141.6, 130.9, 129.3, 123.6,
122.2, 119.3, 104.5, 82.97, 62.17, 60.48. Elemental analysis calculated
for C26H34N4O12: C, 52.52; H, 5.76; N, 9.42%. Found: C, 52.52; H,
5.76; N, 9.39. MALDI-TOF-MS (m/z): 617.21 (calcd), 617.19
(found). The chemical structure of LL-Gly-Azo is shown in Chart 1.
N-p-Phenylazophenyl acetamide-[O-α-D-glucopyranosyl-(1→4)]-D-
gluconamide (MAL-Gly-Azo): maltose was oxidized to yield maltono-
lactone (MAL) in the same manner as LL, and the respective conjugate
was prepared from MAL and Gly-Azo using the method described
above. MAL-Gly-Azo 0.78 g (65.2%) was obtained as a yellowish
crystalline precipitate. Tm: 206 °C with decomposition. 1H NMR
(DMSO-d6): 9.91 (s, 1H), 8.20 (t, 1H), 7.78−7.93 (m, 6H), 7.54−7.60
(m, 3H), 5.77 (d, 1H), 5.70 (d, 1H), 5.00 (d, 1H), 4.95 (d, 1H), 4.53 (t,
1H), 4.45−4.49 (m, 2H), 3.89−4.05 (m, 2H). 13C NMR (DMSO-d6):
172.8, 168.0, 151.9, 147.5, 141.6, 130.9, 129.3, 123.6, 122.2, 119.3, 100.9,
83.1, 69.8, 62.4, 60.5. Elemental analysis calculated for C26H34N4O12: C,
52.52; H, 5.76; N, 9.42%. Found: C, 52.52; H, 5.76; N, 9.39. MALDI-
TOF-MS (m/z): 617.21 (calcd), 617.31 (found). The chemical
structure of MAL-Gly-Azo is shown in Chart 1.
multiple hydroxyl groups in carbohydrate moieties can both
donate and accept protons to form various hydrogen bonds; and
(4) most supramolecular gels are composed of nanofibers,
which allow molecular recognition by proteins. These features
can be used for biological applications such as a lectin array for
saccharide detection,21 wound healing,23 and stimuli-responsive
biomaterials.37,39
In carbohydrate-mediated interactions, the nonreducing ends
of carbohydrate head groups bind with protein receptors.40
Although studies have been carried out on monosaccharide-
based hydrogelators,19−25,39 oligosaccharide head groups18 and
their specificity for protein recognition have seldom been
investigated. We have chosen disaccharide lactones as a hydro-
philic, biofunctional headgroup because they are the simplest
unit to examine the diversity of glycosidic linkages, anomeric
configuration, and positions of hydroxyl groups in bioactive
sugars. In addition, some natural homopolysaccharides are
known to form structures with secondary order. For example,
with regard to glucose (Glc) polymers, amylose (Glc(α1→
4)Glc) forms a helical structure, and cellulose (Glc(β1→4)Glc)
forms sheetlike structures.41,42 Delicate intra- and intermolecular
associations between polysaccharides induce the regular
formation of higher-order structures; as a result, we are interested
in the effects of the noncovalent assembly of saccharide moieties
of sugar-based amphiphiles on the characteristics of ordered
structures formed in supramolecular hydrogels.
In this study, three kinds of low molecular weight amphiphilic
conjugates composed of disaccharide lactones and azobenzene
with glycine as a connecting spacer were synthesized. The
azobenzene chromophore was used as a hydrophobic group,
aiming to allow strong π−π interactions derived from the planar
trans-azobenzene unit as well as reversible association via
photoisomerization. Herein, we show that this supramolecular
self-assembly strategy offers great potential form tailoring
glycocluster formation for specific protein recognitions. Second-
ary and higher-order structures were investigated by spectro-
scopic and microscopic analyses. The ultraviolet (UV)-induced
gel-to-sol transition, lectin binding, and cell adhesion behavior of
the glyco-based structures are also discussed in detail.
EXPERIMENTAL SECTION
■
Chemicals. The following reagents were used as received: lactose,
glycine, β-cyclodextrin, urea, chloroform, methanol (Wako Pure
Chemicals, Osaka, Japan), maltose, cellobiose, di-tert-butyldicarbonate,
4-aminoazobenzene (Sigma-Aldrich), and fluorescein isothiocyanate-
labeled Ricinus communis agglutinin 120 (FITC-RCA120), RCA120,
concanavalin A (ConA), FITC-ConA (Seikagaku Co., Tokyo, Japan).
N-(tert-Butoxycarbonyl)glycine (Boc-Gly) was synthesized by the
conventional method.43 A rat liver cell line (IAR-20) was provided by
the Human Science Research Resources Bank (Osaka, Japan).
William’s medium E, penicillin−streptomycin, and trypsin were
obtained from Invitrogen Corp. (Carlsbad, CA). Collagen gel (Cell
Matrix Type I-P) and phosphate buffered saline (PBS) were purchased
from Nitta Gelatin Inc. (Osaka, Japan), and Nissui Pharmaceutical Co.
Ltd. (Tokyo, Japan), respectively. Tissue culture polystyrene (TCPS)
dishes and plates (24-well) were obtained from Sumitomo Bakelite
Co. Ltd. (Tokyo, Japan). Other chemical reagents were reagent grade
and used without further purification.
N-p-Phenylazophenyl acetamide-[O-β-D-glucopyranosyl-(1→4)]-D-
gluconamide (CL-Gly-Azo): CL-Gly-Azo was synthesized from
cellobionolactone (CL) and Gly-Azo using the procedure described
above. CL-Gly-Azo 0.93 g (78.4%) was obtained as a yellowish
crystalline precipitate. Tm: 211 °C with decomposition. 1H NMR
(DMSO-d6): 10.10 (s, 1H), 8.11 (t, 1H), 7.80−7.91 (m, 6H), 7.53−
7.60 (m, 3H), 5.48 (d, 1H), 5.32 (d, 1H), 5.00 (d, 1H), 4.95 (d, 1H),
4.61 (t, 1H), 4.35−4.41 (m, 2H), 3.97 (m, 2H). 13C NMR (DMSO-d6):
173.0, 167.9, 151.9, 147.5, 141.6, 130.9, 129.3, 123.6, 122.2, 119.3,
104.0, 82.7, 62.2, 61.2. Elemental analysis calculated for C26H34N4O12:
C, 52.52; H, 5.76; N, 9.42%. Found: C, 52.52; H, 5.78; N, 9.39.
MALDI-TOF-MS (m/z): 617.21 (calcd), 617.27 (found). The
chemical structure of CL-Gly-Azo is shown in Chart 1.
General Analysis. Nuclear magnetic resonance (NMR) spectra
were recorded on a JMN-AL400 spectrometer (JEOL Ltd. Tokyo,
Japan) in a solution of DMSO-d6 or CDCl3. Melting point (Tm)
measurement was performed at a heating rate of 5 °C min−1 using a
differential scanning calorimeter (Pyrus 1, PerkinElmer Inc., San Jose,
CA). Elemental analysis was performed with a CHN corder (MT-6,
YANACO Co. Ltd., Kyoto, Japan).
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a
Chart 1. Amphiphilic Disaccharide−Azobenzene Conjugates
a
LL-Gly-Azo: N-p-phenylazophenyl acetamide-[O-β-D-galactopyranosyl-(1→4)]-D-gluconamide; MAL-Gly-Azo: N-p-phenylazophenyl acetamide-
[O-α-D-glucopyranosyl-(1→4)]-D-gluconamide; CL-Gly-Azo: N-p-phenylazophenyl acetamide-[O-β-D-glucopyranosyl-(1→4)]-D-gluconamide.
Gelation Studies. In a typical gelation experiment, disaccharide
amphiphile (10 mg) was put into a sealed sample tube with one of 10
different polar/nonpolar solvents (distilled water, methanol, ethanol,
chloroform, acetone, N,N-dimethylacetamide, N,N-dimethylforma-
mide, dimethyl sulfoxide, n-hexane, and toluene) to result in a con-
centration of 1% (w/v). Likewise, aqueous solutions of β-cyclodextrin
(11 mM), and urea (4 M) or saline solutions, such as physiological
saline or PBS, were subjected to gelation tests. The mixture was first
stirred at ambient temperature and then heated around the
corresponding boiling temperature until the solid material dissolved.
The solution was set aside and allowed to cool to 25 °C for 24 h. The
hydrogelation behavior of all disaccharide derivatives was tested with
a mixture of designated amounts of samples (0.25−5.0 mg) in hot
distilled water (0.5 mL), then allowed to cool, and stabilized at 25 °C
for 24 h. The minimum hydrogelation concentration was determined
using the inverted test tube method.
equilibration at 37 °C, IAR-20 cells (2.0 × 105 cells per well) were
seeded on the top of the gels and then incubated at 37 °C for a
designated time from 3 to 48 h in an incubator containing 5% CO2.
After the removal of suspended (unattached) cells by thorough rinsing
with PBS, adhered cells were detached from the matrix surface by
digestion treatment with 0.05% trypsin in PBS at 37 °C for 15 min,
and then counted by a cell counter. Each cell assay test was repeated
three times.
UV-Induced Sol−Gel Transition of Hydrogels. A high-pressure
mercury lamp (500 W, Ushio, Tokyo, Japan) with the appropriate
glass filter (UV-35+UVD-36C (365 nm), Asahi Technoglass, Tokyo,
Japan) was used as an irradiation source. Hydrogel samples (0.3 wt %)
were sandwiched to a thickness of 0.5 mm using two quartz plates
covered with a photomask and set vertically onto a stand. UV light was
then irradiated vertically on the sample cell through the photomask
with a rectangular window (5 × 10 mm).
UV−vis and Circular Dichroism (CD) Spectroscopy. UV−vis
and CD spectra were obtained using a J-720 CD spectrometer (JASCO,
Japan) with a water-jacketed cylindrical quartz cell with a path length
of 0.1 mm. Each sample (5.0 mg) was suspended in distilled water
(1 mL), heated until it became clear, and then injected into the quartz
cell, which was then carefully sealed. Thermoresponsive UV−vis and
CD spectra were recorded from 20 to 70 °C. Data were collected from
600 to 250 nm, at 0.5 nm intervals with a scan speed of 200 nm min−1
and bandwidth of 1 nm.
LVSEM Observation. Low-voltage scanning electron microscopy
(LVSEM; Wet-SEM, Shimadzu SS-550, Japan) was used to observe
the in situ morphology of the hydrogels. A small piece of each hydrogel
(0.5 wt %) was fixed on the sample holder without any additional
treatment. Measurements were performed under a low vacuum of
20−100 Pa and acceleration voltage of 5−20 kV.
FITC-Labeled Lectin Binding Assay. In this study, two kinds of
FITC-labeled lectins were used for lectin binding assays: one from
Ricinus communis (RCA120) and another from Canavalia ensiformis
(ConA). LL-Gly-Azo and MAL-Gly-Azo were dissolved in PBS solution
and heated until a clear solution was obtained. The resulting hydrogels
were cut into small pieces and then washed 5 times in PBS solution.
Each piece of hydrogel was then immersed in FITC-RCA120 or FITC-
ConA (20 μg mL−1) in PBS solution for 30 s. The gel pieces were
washed with PBS solution at least 3 times and then sandwiched
between two sterile coverslips. Fluorescence microscopic images of the
gel samples were captured using a Nikon 2000 confocal laser scanning
microscope (CLSM; Nikon, Japan). Conjugated FITC moieties were
visualized using excitation and emission wavelengths of 488 and
515 nm, respectively, at room temperature.
Cell Adhesion Test. Rat liver cells (IAR-20, JCRB Cellbank) were
dispersed in a serum-free culture of William’s medium E containing 5%
antigen (penicillin and streptomycin). LL-Gly-Azo (0.5 mL, 0.5% w/v),
and the collagen matrix solution (0.5 mL, 0.3% w/v) dissolved
in William’s medium E was poured into 24-well microtiter plates.
A TCPS 24-well plate was used as a control. After gel formation and
RESULTS AND DISCUSSION
■
Gelation Properties of Azobenzene-Conjugated Car-
bohydrates. All disaccharide derivatives synthesized in this
report were insoluble in nonpolar solvents but readily soluble in
aprotic polar solvents (N,N-dimethylacetamide, N,N-dimethyl-
formamide, and dimethyl sulfoxide). However, the disaccharide
conjugates consisting either of lactose (Gal(β1→4)Glc, LL-Gly-
Azo) or maltose (Glc(α1→4)Glc, MAL-Gly-Azo) dissolved well
in hot water (with or without saline), and clear gels were
successfully formed after standing at room temperature for
several hours. These hydrogels exhibited a thermoreversible sol−
gel transition and were stable in the gel state for more than
10 months. In particular, LL-Gly-Azo possessed excellent hydro-
gelation ability, resulting in obvious water gels at a low con-
centration of 1.0 mg mL−1 (1.68 mM). However, no gelation
occurred in an aqueous solution containing either β-cyclodextrin
or urea. The former is well-known to form an inclusion complex
with azobenzene,45,46 and the latter can eliminate intermolecular
hydrogen bonds between polysaccharides.47 These results
strongly suggest that both the hydrophobic effect of stacking
azobenzene groups and the multiple hydrogen bonds of sugar
moieties made a significant contribution to hydrogel formation.
On the other hand, the cellobiose derivative CL-Gly-Azo
demonstrated different gelation behavior. CL-Gly-Azo did not
form hydrogels up to 10% w/v within 24 h when dissolved in hot
water (just below the boiling point), although it was readily soluble
in water at room temperature, forming a clear gel. Considering the
glycosidic linkage, cellobiose units in cellulose are connected to
each other in a “trans” form; however, several experimental and
theoretical investigations into the conformational preference of
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cellobiose and their derivatives have revealed that isolated and
hydrated cellobiose units adopt a “cis” conformation bound by
inter-ring hydrogen bonds.48,49 These results suggest that water
dynamics could play a critical role in determining the most stable
structure of the cellobiose conjugate. This implies that the thermal
history critically affected the formation of intra- and intermolecular
hydrogen bonds of the cellobiose−azobenzene amphiphile so
that it exhibited irregular gelation behavior. In addition, the CL-
Gly-Azo hydrogel formed was fragile around the gel-to-sol
transition temperature of ca. 30 °C, making this gel unsuitable
for subsequent experiments. The most noteworthy point is that
the difference in gelation behavior is caused by the change in
position of just one hydroxyl group; LL-Gly-Azo is a C4-epimer of
the nonreducing end of CL-Gly-Azo. These results indicate that
the affinity of these compounds for water depends on the rotation
angle of the sugar headgroup, directly affecting their gelation
behavior.
Morphology of Self-Assembled Hydrogels. Hydrogels
consisting either of LL-Gly-Azo or MAL-Gly-Azo are represen-
tative physical gels that form through noncovalent associations
such as hydrophobic and hydrogen bonding interactions. It is
well-known that low molecular weight gelators, namely
nonpolymeric molecular ones, enable construction of nanoscale
suprastructures, such as planar ribbons, helical ribbons,
nanofibrils, and nanosheets. A variety of microscopic techniques
have been used to visualize hydrogels, such as SEM,
transmission electron microscopy, and atomic force micros-
copy.18−20,22−26,31−39 In this research, the gel-state morphology
of LL-Gly-Azo and MAL-Gly-Azo was directly observed using
LVSEM. This method allows us to observe a quasi-actual
hydrogel in a wet state, without any conductive coating.
Therefore, LVSEM images are regarded as being essentially
reflected in situ images of the hydrogels in water. As expected,
these hydrogels formed the entangled structures of nanofibrous
assemblies, as shown in Figure 1. Both gelators appeared to have
investigated previously; e.g., simple micelles can be converted to
ellipsoidal and then cylindrical micelles when their concen-
tration is increased.27,28 Similar phenomena may occur for both
LL-Gly-Azo and MAL-Gly-Azo, which possibly form cylindrical
micellar fibrils in which hydrophobic azobenzene moieties are
directed to the inside of micelles and the hydrophilic
carbohydrate head groups exposed outward are in contact
with surrounding water molecules. In other words, it is
presumed that the sugar moieties self-assemble on the surfaces
of the thin micellar fibrils, so they are present on the surface of
the entangled nanofibers.
Supramolecular Structures of Sugar-Conjugated
Hydrogelators. The geometrically rigid and longitudinal
structures of azobenzene molecules make them ideal mesogens.
Several studies have been carried out investigating the ordered
formation of azobenzene-containing amphiphiles,20,26,33−37 and
their light absorption spectra reflect the secondary structure of
their aggregates. Thus, UV−vis absorption and CD spectral
examinations were performed in solution and gel states to
obtain direct evidence for the stacking of azobenzene moieties
and formation of chiral aggregates in water. Figure 2 shows the
Figure 1. LVSEM images of (a, a′) LL-Gly-Azo and (b, b′) MAL-Gly-
Azo. [Sample] = 0.5% w/v. Scale bars correspond to 10 μm.
Figure 2. Temperature dependence of (a) UV−vis absorbance profiles
and (b) corresponding CD spectra for 0.5% w/v aqueous LL-Gly-Azo.
a twisted structure but did not form well-defined helices.
Interestingly, the LVSEM images of the LL-Gly-Azo hydrogel
(Figure 1a,a′) and MAL-Gly-Azo (Figure 1b,b′) suggested that
numerous thin fibrils aligned in the same direction were
intertwined into bundles of larger fibrils, which conserved the
tertiary structure of the hydrogels. The secondary structure
of aggregates of amphiphilic molecules in water has been
temperature-dependent profiles of the CD spectra and high-
tension (HT) voltage of a solution of LL-Gly-Azo. Upon
heating, the CD intensity gradually decreased, possibly
implying the dissociation of chiral aggregation.50 Moreover,
the CD intensity approached zero above 70 °C, which implies
the absence of regular structures (isotropic state). The HT
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signal, which provides a direct measure of the quantity of light
being produced and passed through the sample, is roughly
proportional to the absorbance. At 75 °C in a sol state, LL-Gly-
Azo exhibited a typical absorption spectrum of a monomeric
azobenzene derivative with a weak n → π* transition at 450 nm
and an intense π → π* transition at 330 nm. Upon cooling to
ambient temperature, the main peak broadened, decreased in
intensity, and exhibited a blue shift (Δλmax = 16 nm) compared
with that for the sol state (Figure 2a). This spectral change
provides evidence for H-type aggregation (card-pack array) of
the chromophore, which has indeed been observed for most
amphiphilic azobenzene derivatives in aqueous solu-
tion.20,33,36,51 A similar shift was also observed for a solution
of MAL-Gly-Azo, although no significant profile was displayed
for a solution of CL-Gly-Azo dissolved in hot water (sol state)
(Figure 3). Such blue shifts of the absorption spectra of fibrillar
Figure 4. CD spectra of 0.5% w/v of (a) LL-Gly-Azo, (b) MAL-Gly-
Azo, and (c) CL-Gly-Azo at 25 °C. All samples were dissolved in hot
water and stored overnight at room temperature.
Because LL-Gly-Azo and MAL-Gly-Azo have different
molecular structures only for the disaccharides, the dominant
factor determining helicity must be the steric structure of the
saccharide headgroup. Assuming that hydrophobic forces have no
influence on the precise formation of hydrogen bonding,56 the
steric position of hydroxyl groups are of great importance to
stabilize the chiral organization through hydrogen bonding
interactions. Furthermore, the intermolecular hydrogen bonds
derived from the amide groups of the Gly linkers as well as π−π
stacking of the azobenzene groups possibly assisted the formation
of microscopic fibrous nanostructures of both hydrogels.
Surface Glyco-Decoration Determined by FITC-La-
beled Lectin Assay. Spectroscopic and microscopic studies
suggested that as-synthesized amphiphilic sugar−azobenzene
conjugates form micelle-like aggregations, with the sugar residues
gathered closely together on the surfaces of the twisted cylindrical
fibers. Hence, a lectin-binding assay was carried out to elucidate
the presence of sugar moieties on the surfaces of the self-
assembled glycohydrogels that the lectin can access to bind.
Lectin has considerable specificity for binding to carbohydrates;
RCA120 can react with β-D-galactosyl residues, and ConA can
interact with α-D-mannosyl and α-D-glucosyl residues.40,57 In this
research, CLSM observation was applied using FITC-labeled
lectins to visualize the sugar assembly on the hydrogels. Figure 5
shows fluorescent CLSM images of hydrogels of LL-Gly-Azo and
MAL-Gly-Azo bound to respective FITC-lectins. A clear fibrous
fluorescent image was observed for the LL-Gly-Azo gel treated
with FITC-RCA120 (Figure 5a), and MAL-Gly-Azo with FITC-
ConA (Figure 5d) was also visualized clearly. Thus, the
nonreducing end group of the carbohydrate structure of each
gelator, either Gal on the LL-Gly-Azo fiber or Glc on the MAL-
Gly-Azo fiber, were effectively recognized by the corresponding
lectins for a very short time within 30 s, in comparison to the
lectin assay system using hydrogelators as proposed by Hamachi
et al., e.g. 30 min.21 It was presumed that the sugar-clustering
effects via azobenzene packing possibly led to such a higher
affinity for the corresponding lectin. By contrast, undesirable
nonspecific binding was not observed because no fluorescent
signal was detected for ConA−LL-Gly-Azo and RCA120−MAL-
Gly-Azo pairs (Figure 5b,c). Consequently, it is expected that
these sugar assemblies could act as sugar−protein recognition
Figure 3. UV−vis absorbance spectra of an aqueous solution of MAL-
Gly-Azo at (a) 25 °C and (b) 50 °C and for a solution of CL-Gly-Azo
at (c) 25 °C and (d) 50 °C. [Sample] = 0.5% w/v.
assembly inside the hydrogels are explained by taking into
consideration the H-type π-stacking of azobenzene chromo-
phores in the conjugate.
Further evidence for nanofiber formation is provided in the
CD spectra of aqueous solutions of LL-Gly-Azo and MAL-Gly-
Azo in a gel state (25 °C) shown in Figure 4. Both amphiphiles
were CD-active, and their zero-crossing point was close to their
absorption maximum (around 330 nm), indicating the CD
Cotton effect was possibly caused by exciton coupling bands
forming between the stacked azobenzene chromophores. The
CD intensity of the same concentration of LL-Gly-Azo and
MAL-Gly-Azo was relatively similar although the polarity was
reversed. This stark contrast means they exhibit opposite helical
chirality in an aggregated state. LL-Gly-Azo gel shows a positive
first Cotton effect and negative second Cotton effect, indicating
a right-handed helical structure, i.e., (R)-chirality. In contrast,
the inverted CD spectrum was observed for the hydrogel of
MAL-Gly-Azo, indicating a left-handed chiral arrangement, i.e.,
(S)-chirality. Several chiral glycohydrogelators20,22,25 and
glycopolymers52−54 with different sugar head groups have
been reported; however, there are few investigations regarding
helical inversion of aggregates induced only by the structure of
the sugar head groups.55
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asialoglycoprotein receptors on the rat liver cell surfaces.61−63
Azobenzene may have some kinds of cytotoxic influences on cell
culture applications; however as-synthesized sugar-conjugated
azobenzenes were densely stacked in a stable gel state to form
the fibrous micelles entangled each other, resulting in negligible
cytotoxicity and good cell adhesion behavior. Therefore, an
increasing trend in the cell attachment on the biocompatible
LL-Gly-Azo hydrogel was attributed to the direct, biological
recognition of Gal residues on the fibrous aggregates. Other
types of biological cells having maltose- and/or glucose-
recognizing receptors would interact with MAL-Gly-Azo gel,
and such a tunable combination is regarded as promising in
biointerface applications.
UV-Induced Sol−Gel Transition Behavior. In an
isotropic state, azobenzene derivatives in solution undergo
photoinduced trans/cis isomerization under UV irradiation,
while cis-to-trans back-isomerization can be activated thermally
or by visible light. The azobenzene moiety in an H-type
aggregate state, however, is known to show remarkable resistance
to UV-induced isomerization. Suppression of photoisomerization
of azobenzene moieties has been observed in tightly packed,
rigid aggregates, so in this case the reversible photoresponse of
azobenzene is limited.26,33−36 Therefore, the hydrogels were
irradiated with high-power UV light to examine the effect of
photoisomerization of azobenzene moieties of sugar conjugates.
The hydrogels were exposed to high-intensity UV light (365 nm,
ca. 10 mW cm−2) through a rectangular pattern in a photomask.
A typical example of UV-exposure testing for the LL-Gly-Azo
gel is shown in Figure 7. Upon irradiation with UV light, only
the exposed area of the gel gradually melts to the bottom of
the sample cell. This observation implies the disruption of
aggregation caused by trans-to-cis photoisomerization of the
azobenzene moieties. Moreover, the resulting sol gelated again
within 12 h under weak visible light (Figure 7) because of back-
isomerization that promotes the reaggregation of azogelator
molecules in a trans form. A similar reversible sol−gel transition
phenomenon was also observed for the hydrogel of MAL-Gly-
Azo, and such a reversible sol−gel transition behavior is expected
to be useful for practical cell culture applications.
Figure 5. Fluorescent images of LL-Gly-Azo hydrogels treated with
(a) FITC-RCA120 and (b) FITC-ConA and MAL-Gly-Azo hydrogel
treated with (c) FITC-RCA120 and (d) FITC-ConA. Each compound
was dissolved in PBS (0.5% w/v, pH 7.2). Scale bars correspond to
10 μm.
sites through their highly packed and intertwined structures,
which results in their high affinity and selectivity for lectins.
Cell Adhesion Assay. The presence of a Gal-specific
receptor on the surfaces of rat hepatocytes is responsible for the
selective uptake of partially deglycosylated glycoproteins.44,58−60
The LL-Gly-Azo hydrogel designed in this study has dense Gal
residues; thus, a cell adhesion assay was carried out to elucidate
the function of the hydrogel as a biointerface. Figure 6 shows
Because photoisomerization of azobenzene moieties occurs
with a large change in molecular geometries, this process is
allowed to proceed only when there is sufficient space present
around the azobenzene units.26,64 In the case of our
disaccharide−azobenzene hydrogelators, the bulky structure of
the sugar headgroup, which can form higher order structure in
the hydrogen bonding network, possibly caused the azobenzene
mesogenic cores to be loosely packed, so they had enough room
to undergo a conformational transition to the cis form. During
photoisomerization of azobenzene, molecular polarity changes
as well as molecular shape. Therefore, two possible mechanisms
can be considered: one is the steric effect of the conjugated
azobenzene, and another is the increasing solubility of the
gelator induced by variation in molecular polarity, resulting in
reduced hydrogen bonding between sugar moieties. As
mentioned above, because these hydrogelators slowly form a
gel again, it is possible to tailor heterogeneous glyco-patterned
gels by the combined use of other hydrogelators with different
sugar head groups under controlled UV irradiation. Such
reversible, tunable glyco-based interfaces are promising for cell
culture applications.
Figure 6. Cell adhesion behavior on (a) 0.5% w/v LL-Gly-Azo gel, (b)
0.3% w/v collagen gel, and (c) TCPS plate following incubation for
1−48 h.
the cell adhesion behavior of rat hepatocytes in serum free
culture. The LL-Gly-Azo gel demonstrated higher cell adhesion
than the collagen gel and TCPS plate. After incubation for 24 h,
cell attachment reached up to 83% on LL-Gly-Azo, although it
was less than 60% and 35% for TCPS and collagen gel,
respectively. As mentioned above, a lectin binding assay with
RCA-120 suggested that the Gal residues were effectively
clustered on the surfaces of the self-assembled fibers, and thus
such initial hepatocyte attachment was possibly dominated by
the clustered Gal residues directly interacting with the
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AUTHOR INFORMATION
■
Corresponding Author
*Tel: + 81 92 642 2993; Fax: + 81 92 642 2993; e-mail: tkitaoka@
agr.kyushu-u.ac.jp.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was financially supported by Grants-in-Aid for
Young Scientists (Start-up: 20880021 to Y.O.; S: 21678002 to
T.K.) from the Ministry of Education, Culture, Sports, Science
and Technology, Japan. LVSEM observation was carried out
using a Shimadzu SS-550 microscope at the Center of
Advanced Instrumental Analysis, Kyushu University, Fukuoka,
Japan. We are indebted to Dr. Yosuke Tsuchiya and Prof. Seiji
Shinkai, Institute of Systems, Information Technologies and
Nanotechnologies, Fukuoka, Japan, for their assistance with CD
spectrometry.
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CONCLUSION
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Novel sugar-decorated nanofibrous hydrogels were successfully
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