Synthesis of Fluorescent Gelators and Direct Observation of Gelation with a Fluorescence Microscope

Article abstract of DOI:10.1002/chem.201603295

Fluorescein-, benzothiazole-, quinoline-, stilbene-, and carbazole-containing fluorescent gelators have been synthesized by connecting gelation-driving segments, including l-isoleucine, l-valine, l-phenylalanine, l-leucine residue, cyclo(l-asparaginyl-l-phenylalanyl), and trans-(1R,2R)-diaminocyclohexane. The emission behaviors of the gelators were investigated, and their gelation abilities studied against 15 solvents. The minimum gel concentration, variable-temperature spectroscopy, transmission electron microscopy, scanning electron microscopy, fluorescence microscopy (FM), and confocal laser scanning microscopy (CLSM) were used to characterize gelation. The intermolecular hydrogen bonding between the N?H and C=O of amide, van der Waals interactions and π–π stacking play important roles in gelation. The colors of emission are related to the fluorescence structures of gelators. Fibrous aggregates characterized by the color of their emission were observed by FM. 3D images are produced by the superposition of images captured by CLSM every 0.1 μm to a settled depth. The 3D images show that the large micrometer-sized aggregates spread out three dimensionally. FM observations of mixed gelators are studied. In the case of gelation, two structurally related gelators with the same gelation-driving segment lead to the gelators build up of the same aggregates through similar hydrogen-bonding patterns. When two gelators with structurally different gelation-driving segments induce gelation, the gelators build up each aggregate through individual hydrogen-bonding patterns. A fluorescent reagent that was incorporated into the aggregates of gels through van der Waals interactions was developed. The addition of this fluorescent reagent enables the successful observation of nonfluorescent gelators’ aggregates by FM.

Full text of DOI:10.1002/chem.201603295

DOI: 10.1002/chem.201603295
Full Paper
&
Supramolecular Chemistry
Synthesis of Fluorescent Gelators and Direct Observation of
Gelation with a Fluorescence Microscope
Kenji Hanabusa,*[a, b] Takuya Ueda,[a] Shingo Takata,[a] and Masahiro Suzuki[a]
Abstract: Fluorescein-, benzothiazole-, quinoline-, stilbene-,
and carbazole-containing fluorescent gelators have been
synthesized by connecting gelation-driving segments, in-
cluding l-isoleucine, l-valine, l-phenylalanine, l-leucine resi-
due, cyclo(l-asparaginyl-l-phenylalanyl), and trans-(1R,2R)-di-
aminocyclohexane. The emission behaviors of the gelators
were investigated, and their gelation abilities studied against
15 solvents. The minimum gel concentration, variable-tem-
perature spectroscopy, transmission electron microscopy,
scanning electron microscopy, fluorescence microscopy (FM),
and confocal laser scanning microscopy (CLSM) were used
to characterize gelation. The intermolecular hydrogen bond-
ing between the NÀH and C=O of amide, van der Waals in-
teractions and p–p stacking play important roles in gelation.
The colors of emission are related to the fluorescence struc-
tures of gelators. Fibrous aggregates characterized by the
color of their emission were observed by FM. 3D images are
produced by the superposition of images captured by CLSM
every 0.1 mm to a settled depth. The 3D images show that
the large micrometer-sized aggregates spread out three di-
mensionally. FM observations of mixed gelators are studied.
In the case of gelation, two structurally related gelators with
the same gelation-driving segment lead to the gelators
build up of the same aggregates through similar hydrogen-
bonding patterns. When two gelators with structurally differ-
ent gelation-driving segments induce gelation, the gelators
build up each aggregate through individual hydrogen-bond-
ing patterns. A fluorescent reagent that was incorporated
into the aggregates of gels through van der Waals interac-
tions was developed. The addition of this fluorescent re-
agent enables the successful observation of nonfluorescent
gelators’ aggregates by FM.
Introduction
tors were rare for the next half century; however, research in-
terest increased again during the first half of the 1990s. With
advances in the area of supramolecular chemistry, several re-
ports on gelators have been published in recent years.[3–21] Al-
though gelators are of special interest to academic researchers,
they also have practical applications. However, the number of
gelators used in practical applications remains small. 1,3:2,4-Di-
benzylidene-d-sorbitol and N-lauroyl-l-glutamate-a,g-bis-n-bu-
tylamide are used as ingredients in fragrances and cosmetics,
12-hydroxystearic acid is used as an edible-oil solidifying
agent, aromatic diureas are used as ingredients for greases,
and aluminum 2-ethylhexanoate, which dissolves at room tem-
perature in hydrocarbon solvents and forms a gel-like viscous
product, is used as a thickener for ink.
In recent years, several researchers have shown considerable
interest in studying gelators that can physically gel water or or-
ganic solvents. Such gelators are of interest because they can
immobilize substantial volumes of solvent. In particular, gela-
tors that can induce gelation when added at masses less than
1 g against 1 L of liquid are called supergelators. Despite the
large number of recent papers about gelators in the literature,
designing a gelator remains a difficult task. Gelation results
from competition between the tendencies for a gelator to dis-
solve in the solvent and the tendencies for it to crystallize.
When a gelator-containing solution is cooled, low-molecular-
weight compounds usually give crystals with different solubili-
ties. The first report regarding gelators was published in 1841,
when Lipowitz described lithium urate hydorgel.[1] The follow-
ing report was published in 1942, when Yamamoto described
the gelator 1,3:2,4-dibenzylidene-d-sorbitol.[2] Papers on gela-
Several electron microscopy techniques such as SEM, TEM,
and AFM have been used to investigate the network structure
of the aggregates formed by gelation. Whereas the use of
SEM, TEM, and AFM techniques in gelator research is routine,
fluorescence microscopy (FM) has not been frequently used
because its use is restricted to fluorescent gelators. With re-
spect to the FM technique, Whitten et al. reported FM images
of stilbene-containing cholesterol in an octanol gel in 1999.[22]
To our knowledge, their work represents the first reported FM
images of gels. The application of FM to gelator research has
been increasing in recent years, resulting in a limited number
of manuscripts.[23–29] Because TEM, SEM, and AFM are limited to
the observation of xerogels, they are not applicable to obser-
[a] Prof. K. Hanabusa, T. Ueda, S. Takata, Prof. M. Suzuki
Interdisciplinary Graduate School of Science and
Technology Shinshu University, Ueda, 386-8567 (Japan)
E-mail: hanaken@shinshu-u.ac.jp
[b] Prof. K. Hanabusa
Division of Frontier Fibers, Institute for Fiber Engineering, ICCER
Shinshu University, Ueda, 386-8567 (Japan)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201603295.
Chem. Eur. J. 2016, 22, 1 – 12
1
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
&
These are not the final page numbers! ÞÞ

Full Paper
vations of wet gels. The observation of real aggregates in wet
gels is obviously critical. FM has an advantage in that it ena-
bles direct observation of a wet gel containing solvent. Anoth-
er advantage of FM is that observations are carried out under
low magnifications of several hundred times. TEM, SEM, and
AFM are effective for observations under high magnifications
of several tens of thousands of times. In the case of low-mag-
nification observations, FM is strikingly different from an elec-
tron microscopy. Considering that the gelation by low-molecu-
lar-weight gelators is a macroscopic expression of molecular
self-assembly, FM is well suited to observe large micrometer-
sized aggregates.
Results and Discussion
Gelation abilities
Fluorescein-containing gelators (2–5) were prepared from fluo-
rescein and the corresponding gelation-driving segments by
Williamson synthesis (Figure 1). Gelators 2, 4, and 5 have l-iso-
leucine, l-valine, and l-phenylalanine residues in their gela-
tion-driving segments, respectively. Meanwhile, gelator 3 is a di-
peptide type, in which l-isoleucyl–l-isoleucyl units are embed-
ded as a gelation-driving segment.
Gelation tests were carried out using the upside-down test-
tube method. The results of gelation tests of 2–5 against
15 solvents are summarized in Table 1. All compounds 2–5
were too soluble in THF and chloroform to act as gelators; by
contrast, they were sparingly soluble in hexane, dodecane, and
acetonitrile. Compound 2 was the best gelator among 2–5
with respect to the number of solvents gelled and the MGCs.
Compound 5, containing l-phenylalanine residue as the gela-
tion-driving segment, gave a precipitate in methanol, 1-propa-
nol, and DMF. This result suggests that l-phenylalanine residue
is not useful as a gelation-driving segment. As will be de-
scribed later, a main driving force for the gelation for our gela-
tors is hydrogen bonding; therefore, we expected compound
3, which includes an l-isoleucyl–l-isoleucyl unit, to exhibit
a strong gelation ability.[30] Contrary to our expectations, com-
pound 3 gave precipitates in cyclohexane, acetone, ethyl ace-
We herein report the synthesis of various fluorescent gela-
tors by connecting typical gelation-driving segments and the
measurement of the gels’ fibrous aggregates by TEM, SEM, and
FM. In particular, we focused on observation using FM. We
clarified the location of each gelator in networks by observing
each emission from the formed gels by mixing two different
gelators. We also mention a simple fluorescent reagent, which
enables the successful observation of aggregates of nonfluor-
escent gelators in FM.
Figure 1. Structure of gelator 1 and fluorescein-containing fluorescent gelators 2–5.
&
&
Chem. Eur. J. 2016, 22, 1 – 12
www.chemeurj.org
2
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

Full Paper
anol and toluene. The gelators 6, 7, 11, and 12 could gel
a wide variety of solvents that range from nonpolar to polar
solvents and include both protic and aprotic ones. The strong
gelation abilities of these gelators suggest that l-isoleucine
and l-leucine residues are effective gelation-driving segments.
Table 1. Gelation tests of fluorescent gelators 2–5 at 258C.[a]
Solvent
2
3
4
5
hexane
I
I
I
I
I
dodecane
cyclohexane
methanol
ethanol
1-propanol
acetone
I
I
P
I
GTL(8)
GO(10)
GO(10)
GO(10)
GO(20)
GO(20)
S
GT(8)
P
P
GO(20)
P
GO(20)
S
GO(20)
P
GO(40)
P
GO(20)
GO(20)
S
GO(20)
GO(20)
GO(20)
P
P
S
ethyl acetate
THF
Variable-temperature spectroscopy
DMF
DMSO
g-butyrolactone
toluene
acetonitrile
chloroform
GTL(8)
GTL(8)
GTL(8)
GO(10)
I
GTL(8)
GTL(8)
GO(20)
GT(8)
I
GO(10)
GO(40)
GO(20)
GT(20)
I
P
VS
GO(40)
GTL(20)
I
S
To ensure the participation of hydrogen bonding in physical
gelation, we collected variable-temperature IR spectra. IR spec-
tra of the DMSO gel (8 mgmLÀ1) of 2 collected over the range
from 30 to 808C is shown in Figure 3; the nNÀH of amide I,
nCÀH of methylene, nC=O of amide I, and dNÀH of amide II
are evident in these spectra. The DMSO gel at 308C was gradu-
ally changed to the solution upon heating; specifically, the gel
almost collapsed at 508C and the isotropic solution was visual-
ly observed to have formed at temperatures above 608C. The
nNÀH (amide I), nCÀH (methylene), and nC=O (amide I) peaks
were shifted by 7, 2, and 9 cmÀ1 toward higher frequencies
with increasing temperature, respectively. By contrast, the peak
of dNÀH (amide II) was shifted toward lower frequencies, from
1595 to 1598 cmÀ1, with increasing temperature. These ob-
served frequency shifts indicate the transition from an associa-
tion-type gel to a nonassociation-type gel with increasing tem-
perature. These results led us to conclude that the hydrogen
bonding between NÀH and C=O of amide and van der Waals
interactions are the main driving forces for molecular aggrega-
tion to create a network structure for gel formation.
S
S
S
[a] GT: transparent gel, GTL: translucent gel, GO: opaque gel, S: soluble, I:
almost insoluble, VS: viscous solution. The values indicate the minimum
gel concentrations at 258C; the units are gLÀ1 (gelator per solvent).
tate, and THF instead of gels. The crystallinity of 3 may arise
from the absence of an appropriate hydrophile-lipophile bal-
ance. The balance between gelation and crystallization is clear-
ly delicate.
Fluorescent gelators (6–12) were prepared by Williamson
synthesis or by a coupling reaction with the corresponding ge-
lation-driving segments (Figure 2). The results of gelation tests
of 6–12 are summarized in Table 2. The compounds 6, 9, and
10 have benzothiazole as fluorescent segments. Compounds 7
and 8 possess quinoline and stilbene, respectively. Both 11 and
12 have carbazole as fluorescent segments. All compounds in
Table 2 were miscible in THF and chloroform. By contrast, they
were almost insoluble in hexane and precipitated as crystals
from acetone and acetonitrile. The gelators 9 and 10, which in-
cluded trans-(1R,2R)-diaminocyclohexane and cyclo(l-aspara-
ginyl-l-phenylalanyl), could gel certain solvents such as 1-prop-
To study the existence of p–p stacking among fluorescein
segments, we collected visible spectra of the DMSO gel
(4 mgmLÀ1) of 2 at 30 and 808C. The maximum absorption
wavelength of 462 nm at 808C was blueshifted to 460 nm at
308C. This blueshift suggests the existence of p–p stacking
similar to that of a J-aggregate in the DMSO gel of 2.
Table 2. Gelation tests of fluorescent gelators 6–12 at 258C.[a]
Solvent
6
7
8
9
10
11
12
hexane
I
I
I
I
I
P
P
I
I
P
P
I
GTL(10)
GTL(20)
GTL(20)
GO(10)
GO(10)
GO(20)
P
dodecane
cyclohexane
methanol
ethanol
1-propanol
acetone
GO(20)
GT(20)
GO(40)
GO(40)
GO(40)
P
GO(20)
GT(20)
GO(20)
GO(20)
GO(40)
P
GO(40)
I
P
GO(40)
GO(40)
P
GO(20)
GT(20)
GO(20)
GO(20)
GTL(20)
P
P
GO(40)
GO(40)
S
GO(40)
P
ethyl acetate
THF
GO(40)
S
GO (40)
S
P
S
GO(10)
S
GO(40)
S
GO(20)
S
GTL(20)
S
DMF
DMSO
g-butyrolactone
toluene
acetonitrile
chloroform
GO(40)
GO(40)
GO(40)
GT(40)
P
GO(10)
GO(10)
GO(10)
GT(20)
P
GO(40)
GO(40)
GO(40)
GTL(10)
I
P
S
S
S
GO(10)
GO(10)
GO(10)
GT(40)
P
GO(20)
GO(20)
GO(10)
S
GO(10)
S
GO(40)
P
GT(40)
P
S
GO(40)
P
S
S
S
S
S
[a] GT: transparent gel, GTL: translucent gel, GO: opaque gel, S: soluble, I: almost insoluble. The values indicate the minimum gel concentrations at 258C;
the units are gLÀ1 (gelator per solvent).
Chem. Eur. J. 2016, 22, 1 – 12
www.chemeurj.org
3
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
&
These are not the final page numbers! ÞÞ

Full Paper
Figure 2. Structure of fluorescent gelators 6–12 and compounds 13 and 14.
TEM studies
fective construction. The xerogel of 9 in ethyl acetate loose gel
exhibited thick and short fibers with widths of several hun-
dreds of nanometers. Notably, the ethyl acetate gel of 9 pre-
cipitated as crystals after standing for several hours. The ethyl
acetate gel instability of 9 is attributed to its thick and short
fibers. Xerogel of 10 in ethanol loose gel also contained thick
and short fibers the entanglement of which was gentle. The
relatively high MGC (40 mgmLÀ1) of 10 against ethanol may
result from the sparsely juxtaposed and interlocked fibers.
Figure 4 shows TEM images of xerogels of fluorescent gelators
2, 3, 4, 6, 7, 9, and 10, which were prepared from the corre-
sponding loose gels at a concentration of 2 mgmLÀ1. The xero-
gels prepared from DMSO loose gels of 2 and 3 exhibited
steric networks of juxtaposed, interlocked, and close fibers
with nearly homogeneous diameters. Given that the MGCs of
2 and 3 for DMSO were very low (8 mgmLÀ1), the homogene-
ous dense fibers are a consequence of the steric networks’ ef-
&
&
Chem. Eur. J. 2016, 22, 1 – 12
www.chemeurj.org
4
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

Full Paper
Figure 3. VT-IR spectra of 2 in DMSO gel at 30–808C. [2]=8 mgmLÀ1
.
Figure 4. TEM images of xerogels: a) DMSO gel of 2 (2 mgmLÀ1), b) DMSO gel of 3 (2 mgmLÀ1), c) DMSO gel of 4 (2 mgmLÀ1), d) EtOH gel of 6 (2 mgmLÀ1),
e) EtOH gel of 7 (2 mgmLÀ1), f) ethyl acetate gel of 9 (2 mgmLÀ1), g) EtOH gel of 10 (2 mgmLÀ1).
Chem. Eur. J. 2016, 22, 1 – 12
www.chemeurj.org
5
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
&
These are not the final page numbers! ÞÞ

Full Paper
FM studies
For observations by TEM and SEM, dry samples must be pre-
pared. However, the observation of real aggregates in wet
gels, excluding xerogels, is important. FM has an advantage
that we can directly observe a wet gel containing a solvent.
Figure 5 shows FM 2D images of fluorescent gelators (3, 5, 7,
8, 9, and 11). Figure 5a and b are the FM images of the DMSO
gel of fluorescein-containing 3 and the ethanol gel of fluores-
cein-containing 5, the images of which are characterized by
yellow light emission. Figure 5c, d, e, and f show FM images of
the ethanol gel of 7, toluene gel of 8, ethyl acetate gel of 9,
and the ethanol gel of 11, respectively. The quinoline-contain-
ing 7, stilbene-containing 8, benzothiazole-containing 9, and
carbazole-containing 11 emitted blue, green, blue, and light-
blue light, respectively. The emissions are generated from the
corresponding fluorescent segments in the aggregates. The
dark areas indicate locations at which solvent molecules are
trapped. The observed micrometer-sized aggregates in FM are
likely more realistic images of gels compared to TEM images of
xerogels. Patterns of entanglement among fibrous aggregates
were classified either as a bundle form or as a web form re-
sembling the webs spun by spiders, depending on the gelators
and solvents. For example, the images in Figure 5a, b, and d
show bundle forms, whereas those in Figure 5c, e, and f show
web forms. In particular, the structures of gelation-driving seg-
ments may play an important role in the patterns of entangle-
ment. The present results are considered sufficient to demon-
strate the usefulness of FM in characterizing fluorescent gela-
tors.
Figure 5. FM images of gels: a) 3 in DMSO gel (8 mgmLÀ1), b) 5 in EtOH gel
(40 mgmLÀ1), c) 7 in EtOH gel (40 mgmLÀ1), d) 8 in toluene gel
(10 mgmLÀ1), e) 9 in ethyl acetate gel (10 mgmLÀ1), f) 11 in EtOH gel
(40 mgmLÀ1).
Morphological changes during cooling and heating process-
es were also studied by FM. Figure 6 shows FM images of the
DMSO gel (4 mgmLÀ1) of fluorescein-containing 2 upon cool-
ing. The image immediately collected after the samples were
heated showed yellow light emitted obscurely as a whole (Fig-
ure 6a), where no aggregates could be observed. This lack of
aggregates is why the sample immediately after heating is still
a homogeneous solution. When the sample was rapidly cooled
to room temperature, partial fibrous aggregates appeared 8 s
later (Figure 6b). The aggregates gradually grew and spread
out approximately 3 min later (Figure 6c). The aggregates en-
tangled and radially expanded; they were clearly observed ap-
proximately 9 min later (Figure 6d), by which time the gelation
had completed. By contrast, during the heating process, the
aggregates suddenly drifted away 80 s later and the flowing
aggregates disappeared 210 s later. This observation indicates
that the collapse of the gels begins with disentanglement.
Figure 6. FM images of 2 in DMSO (4 mgmLÀ1): a) immediately after heating,
b) after standing for 8 s, c) after standing for 170 s, d) after standing for
9 min.
are shown in Figure 7b and c, respectively. The image at
point 1 is characterized by the entanglement of many fibrous
aggregates; however, this image is partially out of focus and
was affected by the emission from 2 dissolved in DMSO (Fig-
ure 7b). By contrast, fibrous aggregates were clearly observed
at point 2 (Figure 7c).
Observation of 3D structure by CLSM
The 3D structure of the DMSO gel (4 mgmLÀ1) of fluorescein-
containing 2 was observed by CLSM. The elucidation of the
network structure in gels with CLSM techniques as described
here is, as far as we know, unprecedented. The side-view of
Prꢁparat is shown in Figure 7a. We focused on points 1 and 2,
which have different thicknesses, and carried out 3D observa-
tions. The 2D images of the arbitrary depth at points 1 and 2
We next constructed 3D images of points 1 and 2. This tech-
nique is the similar to the one when CLSM is used for cell
imaging, called z-stacking.[31,32] Images were collected every
0.1 mm to a depth of 80 mm at point 1, resulting in a total of
&
&
Chem. Eur. J. 2016, 22, 1 – 12
www.chemeurj.org
6
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

Full Paper
The obtained clear 3D image is attributed to the exclusion
emissions outside the observation range. Given the combined
CLSM results, we concluded that the large micrometer-size ag-
gregates, which were observed in the 2D FM images, spread
out three-dimensionally and that the resulting 3D networks
trap solvents in space, finally inducing gelation.
FM images of two-component gelators
When gels are formed by the addition of structurally different
gelators, a question arises as to whether they are disorderly in-
corporated into fibrous aggregates or form their respective ag-
gregates separately. To answer this question, we focused on
gels formed by the combination of two gelators. One of the
two-component gelator systems is a combination of gelator
1 and carbazole-containing gelator 11, which includes the
same gelation-driving segment of l-isoleucine. The other
system is a combination of fluorescein-containing gelator 2
and benzothiazole-containing gelator 10, which includes sub-
stantially different gelation-driving segments.
Figure 7. Illustration of Prꢁparat for CSLM and images: a) Prꢁparat indication
point 1 and 2, b) CSLM image of DMSO gel of 2 at point 1, c) CSLM image of
DMSO gel of 2 at point 2.
Figure S1a, in the Supporting Information, shows the FM 2D
image of an ethanol solution (4 mgmLÀ1) of 11, and Figure S1b
shows an ethanol gel prepared from the mixture of
1 (36 mgmLÀ1) and 11 (4 mgmLÀ1). The FM 2D image of the
ethanol gel (40 mgmLÀ1) of 11 was characterized by light-blue
emission from bundled aggregates (Figure 5e). Given the lack
of a fluorescent segment in 1, the FM image of 1 was a dark
field. The ethanol solution (4 mgmLÀ1) of 11 also gave an
almost dark-field image because the gel failed to form when
the concentration of 11 was 4 mgmLÀ1, which is substantially
less than the MGC (Figure S1a). Aggregates that emitted light-
blue light were observed in the FM image of the ethanol gel
of 1 containing a small amount of 11 (Figure S1b). Because the
gelator 1 emits no light, we assumed that the fluorescent gela-
tor 11 was disorderly incorporated into fibrous aggregates of
1. When gels are formed by two structurally related gelators
with the same gelation-driving segment, the gelators conceiva-
bly build up the same aggregates through a similar hydrogen-
bonding pattern.
800 images, and a total of 90 images were taken at 0.1 mm in-
tervals to a depth of 9 mm at point 2. The obtained 3D images
of a 200ꢂ200 mm area are shown in Figure 8. In the 3D image
at point 1, numerous dense fibrous aggregates were observed
and the emission was intense (Figure 8a). Even though the 2D
images were collected every 0.1 mm, the obtained 3D image at
point 1 was blurred. The result is best interpreted by the exis-
tence of numerous fibrous aggregates that spread out three-
dimensionally over a depth of 80 mm at point 1. Consequently,
the emission from fluorescein outside the observation range
was also detected. At point 2, where images were taken every
0.1 mm to a depth of 9 mm, the 3D image was clear (Figure 8b).
We next studied gels prepared from the mixture of fluores-
cein-containing gelator 2 and benzothiazole-containing gelator
10, which include l-isoleucine residue and cyclo(l-asparaginyl-
l-phenylalanyl) as gelation-driving segments, respectively. The
FM image in Figure 9a was due to the UV excitation of benzo-
thiazole-containing gelator 10. Although the fluorescein-con-
taining gelator 2 simultaneously emits yellow under UV excita-
tion, this yellow emission was cut off by the fluorescence filter
in the case of Figure 9a. Figure 9b shows the image obtained
by G excitation, which was obtained by observing the location
of the same Prꢁparat of Figure 9a. Because no fluorescence
was observed from the benzothiazole-containing gelator 10
under G excitation, the yellow emission in Figure 9b was
caused from the fluorescein-containing gelator 2. Figure 9c
was constructed by overlaying two images. Figure 9c indicates
that the aggregates, which emitted light-blue and yellow light,
are separately present from each other. That is, gelators 2 and
10 separately form their respective network structures in the
Figure 8. The 3D images of a 200 mm square prepared by superposition of
CSLM images at the point 1 (above) and point 2 (below).
Chem. Eur. J. 2016, 22, 1 – 12
www.chemeurj.org
7
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
&
These are not the final page numbers! ÞÞ

Full Paper
Figure 10. Illustration of the gelation process by two-component gelators:
a) gelation process by two structurally related gelators including the same
gelation-driving segment, b) gelation process by two gelators with structur-
ally different gelation-driving segments.
Figure 9. FM images of EtOH gels from two-component gelators 2
(10 mgmLÀ1) and 10 (10 mgmLÀ1): a) image by UV excitation, b) image by G
excitation, c) image constructed by overlaying two images.
gel. We conclude that when two gelators with structurally dif-
ferent gelation-driving segments cause gelation, the gelators
build up each aggregate through individual hydrogen-bonding
patterns.
cent reagent 13. Notably, gelator 14 could gel ordinary sol-
vents, but not emit under UV excitation. Figure 11 shows CLSM
images of a 1-propanol gel (20 mgmLÀ1) and a DMSO gel
(10 mgmLÀ1) of 14; these gels contain 1 mg of fluorescent re-
agent 13. The 1-propanol and DMSO gels of 14 exhibit steric
networks of close fibers and thick fibers that emit yellow light
because of the presence of 13. Neither clusters nor precipitates
of 13 were observed in the images. Because fluorescent re-
agent 13 has no hydrogen-bonding sites, we assumed that 13
would be incorporated into the aggregates of 14 through van
der Waals interactions among long alkyl groups. Compound
13 will be very useful as a fluorescent reagent for FM observa-
tions because most gelators have long alkyl chains.
The fact that structurally different gelators build up each mi-
crometer-sized aggregate in gels was also confirmed by FE-
SEM and TEM (Figure S2, Supporting Information). FE-SEM and
TEM images of the benzothiazole-containing gelator 10 were
characterized by aggregates comprising short fibers (Fig-
ure S2a and b), whereas images of the fluorescein-containing
gelator 2 were characterized by bundle aggregates (Figure S2c
and d). In the FE-SEM and TEM images of two-component xe-
rogels prepared from the mixture of 2 and 10, different aggre-
gates, each with its own aggregate characteristics, were ob-
served (Figure S2e and f). These results suggest that gelators 2
and 10 separately form their respective nanometer-sized ag-
gregates in the early stage of gelation and then simultaneously
but separately grow into their respective aggregates, which
are the large micrometer-sized aggregates observed by FM.
On the basis of the results of gelation by two-component
gelators, we illustrate the gelation processes as shown in
Figure 10. In the case of gelation by two structurally related
gelators including the same gelation-driving segment, the ge-
lators build up the same aggregates through similar hydrogen-
bonding patterns (Figure 10a). When two gelators with struc-
turally different gelation-driving segments cause gelation, the
gelators build up each aggregate through individual hydro-
gen-bonding patterns (Figure 10b).
Figure 11. CSLM images of 1-PrOH gel (20 mgmLÀ1) and DMSO gel
(10 mgmLÀ1) of 14, which are contain 1 mg of fluorescent reagent 13.
Conclusions
We prepared fluorescein, benzothiazole, quinoline, stilbene,
and carbazole-containing fluorescent gelators by connecting
typical gelation-driving segments. The gelators, including l-iso-
leucine or l-leucine residue as gelation-driving segments,
showed strong gelation abilities. Close fibers with nearly ho-
mogeneous diameters were observed in TEM images of gels
formed by gelators with low MGCs. Variable-temperature IR
spectra suggested that the hydrogen bonding between the
NÀH and C=O of amide and van der Waals interactions are im-
Fluorescent reagent for FM
Gelators do not always include fluorescence segments, and FM
is not applicable to nonfluorescent gelators. To observe aggre-
gates of nonfluorescent gelators using FM, we prepared fluo-
rescent reagent 13. That is, the fluorescent reagent that we
propose here is thought to act like osmium tetroxide as a nega-
tive staining reagent in the preparation of TEM samples. We
collected FM images of gelator 14 in the presence of fluores-
&
&
Chem. Eur. J. 2016, 22, 1 – 12
www.chemeurj.org
8
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

Full Paper
leucylaminooctadecane. A solution of 9.92 g (20 mmol) of l-iso-
leucyl-l-isoleucylaminooctadecane and 5.4 mL (40 mmol) of trie-
thylamine in 300 mL of dry THF was cooled in an ice/water bath,
and then 6.24 g (22 mmol) of 11-bromoundecanoyl chloride was
added drop-by-drop. The matter was stirred for 1 h in ice/water
bath, followed by for 4 h at room temperature. A filtrate without
NEt3/HCl salt was evaporated and recrystallized from 1-propanol.
N-11-Bromoundecanoyl-l-isoleucyl-l-isoleucylaminooctadecane was
obtained in a yield of 14.0 g (96%). Fluorescent gelator 3 was ob-
tained from a mixture of 0.66 g (2.0 mmol) of fluorescein, 0.44 g
(3.2 mmol) of potassium carbonate, 0.33 g (2.0 mmol) of potassium
iodide, 3.21 g (4.4 mmol) of N-11-bromoundecanoyl-l-isoleucyl-l-
isoleucylaminooctadecane, and 50 mL of dry DMF by the similar
procedure described in gelator 2. An obtained crude product was
purified by column chromatography on alumina (elution with
chloroform/methanol=9:1; Rf =0.9). The fluorescent gelator 3 was
obtained by recrystallization from ethanol/ether in a yield of 2.26 g
(69%). IR (KBr): n˜ =3284 (nNH amide A), 1725 (nC=O lactone), 1633
(nC=O amide I), 1547 cmÀ1 (dN-H amide II); elemental analysis
calcd (%) for C102H170N6O11: C 73.96, H 10.34, N 5.07; found: C 73.52,
H 10.77, N 5.38; excitation maximum: 382 nm; emission maximum:
546 nm (chloroform).
portant main driving forces of gelation. By FM observation, we
directly observed the fibrous aggregates in wet gel containing
solvents. The 3D images, which were produced by the super-
position of images taken by CLSM every 0.1 mm to a settled
depth, suggested that the large micrometer-sized aggregates
spread out three-dimensionally and that the resulting 3D net-
work traps solvents in the space, finally inducing gelation. FM
observations of mixed gelators gave two conclusions. When
gels are formed by two structurally related gelators with the
same gelation-driving segment, the gelators build up the same
aggregates through similar hydrogen-bonding patterns. By
contrast, when two gelators including structurally different ge-
lation-driving segments cause gelation, the gelators build up
each aggregate through individual hydrogen-bonding pat-
terns. We developed a simple fluorescent reagent, that is, a flu-
orescein derivative with two long alkyl chains. The addition of
this fluorescent reagent enabled the successful observation of
aggregates of nonfluorescent gelators by FM. Because this flu-
orescent reagent is incorporated into the aggregates of gels
through van der Waals interactions, it may be useful as a fluo-
rescent reagent for FM observations.
Fluorescent gelator 4: A solution of 7.37 g (20 mmol) of l-valyla-
minooctadecane[34] and 5.4 mL (40 mmol) of triethylamine in
400 mL of dry THF was cooled in an ice/water bath, and then
6.24 g (22 mmol) of 11-bromoundecanoyl chloride was added
drop-by-drop. The matter was stirred for 1 h in an ice/water bath,
followed by for 4 h at room temperature. The recrystallization from
ligroin gave N-11-bromoundecanoyl-l-valyaminooctadecane in
a yield of 10.49 g (85%). Fluorescent gelator 4 was obtained from
a mixture of 0.66 g (2.0 mmol) of fluorescein, 0.44 g (3.2 mmol) of
potassium carbonate, 0.33 g (2.0 mmol) of potassium iodide, 2.71 g
(4.4 mmol) of N-11-bromoundecanoyl-l-valylaminooctadecane, and
50 mL of dry DMF by the similar procedure described in gelator 2.
An obtained crude product was purified by column chromatogra-
phy on alumina (elution with chloroform/methanol=9:1; Rf =0.9).
The fluorescent gelator 4 was obtained by recrystallization from
ethanol/ether in a yield of 1.85 g (66%). IR (KBr, cmÀ1): n˜ =3288
(nNH amide A), 1725 (nC=O lactone), 1634 (nC=O amide I),
1545 cmÀ1 (dNÀH amide II); elemental analysis calcd (%) for
C89H146N4O9: C 75.38, H 10.52, N 3.95; found: C 75.12, H 10.65, N
4.05; excitation maximum: 380 nm; emission maximum: 546 nm
(chloroform).
Experimental Section
Instrumentation
Elemental analysis was performed with a Perkin–Elmer 240B ana-
lyzer. IR spectra were recorded on a Jasco FTIR-7300 spectrometer
by using a KBr plate. TEM and field-emission SEM (FE-SEM) were
done with a JEOL JEM-SS and a Hitachi S-5000, respectively.
1H NMR spectra were taken in CDCl3 on a Bruker AVANCE 400 spec-
trometer and the chemical shifts were recorded in ppm (d) down-
field from TMS. UV/Vis and fluorescence spectra were recorded on
a Jasco V-570UV/VIS/NIR and a Jasco FP-6300. FM and confocal
laser scanning microscopy (CLSM) were performed with a Carl
Zeiss Axio Imager M1 and an Olympus FV1000-D, respectively.
Synthesis of fluorescent gelators
Fluorescent gelator 2: A mixture of 0.66 g (2.0 mmol) of fluores-
cein, 0.44 g (3.2 mmol) of potassium carbonate, 0.33 g (2.0 mmol)
of potassium iodide, 2.77 g (4.4 mmol) of N-11-bromoundecanoyl-
l-isoleucylaminooctadecane (1),[33] and 70 mL of dry DMF was
stirred at 508C overnight under an argon atmosphere. The mixture
was poured to iced water and the precipitated matter was filtered
off, washed with water, and dried. The crude product obtained
was purified by column chromatography on alumina (elution with
chloroform/methanol=9:1; Rf =0.9). Fluorescent gelator 2 was ob-
tained by recrystallization from ethyl acetate in a yield of 2.03 g
(71%). IR (KBr): n˜ =3288 (nNH amide A), 1727 (nC=O lactone),
1637 cmÀ1 (nC=O amide I), 1545 cmÀ1 (dNÀH amide II); elemental
analysis calcd (%) for C90H148N4O9: calcd for C 75.58, H 10.43, N
3.92; found: C 75.20, H 10.57, N 3.79; excitation maximum:
381 nm; emission maximum: 547 nm (chloroform).
Fluorescent gelator 5: Fluorescent gelator 5 was obtained from
a mixture of 0.66 g (2.0 mmol) of fluorescein, 0.44 g (3.2 mmol) of
potassium carbonate, 0.33 g (2.0 mmol) of potassium iodide, 2.92 g
(4.4 mmol) of N-11-bromoundecanoyl-l-phenylalanylaminooctade-
cane, and 50 mL of dry DMF by a similar procedure as that de-
scribed for gelator 2. An obtained crude product was purified by
column chromatography on alumina (elution with chloroform/
methanol=94:4; Rf =0.9). The fluorescent gelator 5 was obtained
by recrystallization from ethanol/ether in a yield of 2.00 g (67%). IR
(KBr, cmÀ1): n˜ =3281 (nNH amide A), 1722 (nC=O lactone), 1640
(nC=O amide I), 1548 cmÀ1 (dNÀH amide II); elemental analysis
calcd (%) for C97H146N4O9: C 76.94, H 9.85, N 3.70; found: C 76.51 H
9.97, N 3.65; excitation maximum: 381 nm; emission maximum:
546 nm (chloroform).
Fluorescent gelator 3: N-Benzyloxycarbonyl-l-isoleucyl-L-isoleucy-
laminooctadecane[30] (31.5 g, 50 mmol) was hydrogenated in the
presence of Pd/C in 300 mL of 1-propanol for 5 h. After confirming
the complete removal of protecting group by TLC, the solution
was filtered off. The filtrate was evaporated and recrystallization
from 200 mL of ligroin provided 22.3 g (90%) of l-isoleucyl-l-iso-
Fluorescent gelator 6: A mixture of 0.91 g (4.0 mmol) of 2-(2-hy-
droxyphenyl)benzothiazole, 0.88 g (6.4 mmol) of potassium carbon-
ate, 0.66 g (4.0 mmol) of potassium iodide, 2.77 g (4.4 mmol) of 1,
Chem. Eur. J. 2016, 22, 1 – 12
www.chemeurj.org
9
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
&
These are not the final page numbers! ÞÞ

Full Paper
and 40 mL of dry DMF was stirred at 508C overnight under an
argon atmosphere. The mixture was poured into iced water and
the precipitated matter was filtered off, washed with water, and
dried. A crude product obtained by recrystallization from ethanol
was purified by column chromatography on alumina (elution with
chloroform; Rf =0.85). The fluorescent gelator 6 was obtained by
recrystallization from ethanol in a yield of 2.11 g (68%). IR (KBr):
n˜ =3288 (nNH amide A), 1634 (nC=O amide I), 1542 (dNÀH ami-
de II), 753 cmÀ1 (benzothiazole); 1H NMR (400 Hz, CDCl3, TMS,
258C): d=5.78 (t, J=5.8 Hz, 1H; CONHCH), 6.00 (d, J=8.8 Hz, 1H;
CHCONH), 7.04 (m, 2H; 3-PhH, 5-PhH), 7.35–7.50 (m, 3H; 4-PhH, 5,
6-benzothiazoleH), 7.93 (d, J=8.5 Hz, 1H; 6-PhH), 8.07 (d, J=
8.8 Hz, 1H; 4-benzothiazoleH), 8.54 ppm (d, J=9.6 Hz, 1H; 7-ben-
zothiazoleH); elemental analysis calcd (%) for C48H77N3O3S: C 74.27,
H 10.00, N 5.41; found: C 73.80, H 10.54, N 6.09; excitation maxi-
mum: 354 nm; emission maximum: 417 nm (ethanol).
propanol and cooled to room temperature. The formed gel was
ground by mechanical stirrer and filtered, followed by drying. 6-
Bromohexyloxy-cyclo(l-asparaginyl-l-phenylalanyl) was obtained in
a yield of 15.14 g (89%). A mixture of 0.91 g (4.0 mmol) of 2-(2-hy-
droxyphenyl)benzothiazole, 0.88 g (6.4 mmol) of potassium carbon-
ate, 0.66 g (4.0 mmol) of potassium iodide, 1.70 g (4.0 mmol) of 6-
bromohexyloxy-cyclo(l-asparaginyl-l-phenylalanyl), and 40 mL of
dry DMF was stirred at 508C overnight under an argon atmos-
phere. The mixture was poured to iced water and the precipitated
matter was filtered off, washed with water, and dried. Recrystalliza-
tion from ethyl acetate gave 1.92 g (84%) of 10. Rf =0.9 (chloro-
form/methanol=9:1, silica gel); IR (KBr): n˜ =1739 (nC=O ester),
1673 (nC=O amide I), 755 cmÀ1 (benzothiazole); elemental analysis
calcd (%) for C32H33N3O5S: C 67.23, H 5.82, N 7.35; found: C 65.92,
H 5.95, N 7.33; excitation maximum: 351 nm; emission maximum:
434 nm (toluene).
Fluorescent gelator 7: A mixture of 0.29 g (2.0 mmol) of 8-hy-
droxyquinoline, 0.44 g (3.2 mmol) of potassium carbonate, 0.33 g
(2.0 mmol) of potassium iodide, 1.39 g (2.2 mmol) of 1, and 20 mL
of dry DMF was stirred at 508C overnight under an argon atmos-
phere. The mixture was poured into iced water and the precipitat-
ed matter was filtered off, washed with water, and dried. A crude
product obtained by recrystallization from ethanol was purified by
column chromatography on alumina (elution with chloroform; Rf =
0.7). The fluorescent gelator 7 was obtained by recrystallization
from methanol in a yield of 0.90 g (63%). IR (KBr): n˜ =3287 (nNH
amide A), 1634 (nC=O amide I), 1541 (dNÀH amide II), 820, 789,
745 cmÀ1 (quinoline); elemental analysis calcd (%) for C44H75N3O3: C
76.14, H 10.89, N 6.05; found: C 75.91, H 10.96, N 6.18; excitation
maximum: 358 nm; emission maximum: 415 nm (ethanol).
Fluorescent gelator 11: To a solution of 13.54 g (81 mmol) of car-
bazole and 1.94 g (81 mmol) of NaH in 90 mL of dry DMF, was
added 22.61 g (81 mmol) of methyl 11-bromoundecanoate and
0.083 g (0.5 mmol) of potassium iodide. The resulting mixture was
stirred at 808C overnight under an argon atmosphere. The mixture
was poured to iced water and the precipitated matter was filtered
off and washed with water. A wet crude product was hydrolyzed in
400 mL of a mixture of acetone, methanol, and water (5:2:3) in-
cluding 6.48 g (162 mmol) of NaOH at 408C overnight. After neu-
tralization by hydrochloric acid, a precipitate was filtered off and
dried. A crude product was recrystallization from 100 mL of metha-
nol. The obtained product was further purified by column chroma-
tography on silica gel (elution with chloroform; Rf =0.16). Recrystal-
lization from methanol gave 11-(9H-carbazol-9-yl)undecanoic acid
in a yield of 14.53 g (51%). A mixture of 7.03 g (20 mmol) of 11-
(9H-carbazol-9-yl)undecanoic acid, 7.65 g (20 mmol) of l-isoleucyla-
minooctadecane, 2.28 g (22 mmol) of DIPC, 2.44 g (20 mmol) of
DMAP (4-dimethylaminopyridine), and 100 mL of dichloromethane
was refluxed overnight. Chloroform (100 mL) was added to the re-
action mixture and an insoluble matter was removed. The resulting
filtrate was washed with 100 mL of 1m hydrochloric acid, followed
by 100 mL of water and dried. After evaporating, recrystallization
from methanol gave 10.04 g (70%) of 11. Rf =0.7 (chloroform, alu-
mina); IR (KBr): n˜ =3289 (nNH amide A), 1634 (nC=O amide I), 748,
719 cmÀ1 (carbazole); elemental analysis calcd (%) for C47H77N3O2: C
78.83, H 10.84, N 5.87; found: C 77.93, H 11.13, N 5.71; excitation
maximum: 405 nm; emission maximum: 482 nm (chloroform).
Fluorescent gelator 8: This compound was obtained by the reac-
tion of 1 and trans-4-(4-aminostyrly)benzonitrile according to our
previous paper.[33]
Fluorescent gelator 9: A mixture of 1.00 g (4.4 mmol) of 2-(2-hy-
droxyphenyl)benzothiazole, 0.88 g (6.4 mmol) of potassium carbon-
ate, 0.66 g (4.0 mmol) of potassium iodide, 1.22 g (2.0 mmol) of
trans-(1R,2R)-bis(11-bromoundecanoylamino)cyclohexane,[35]
and
50 mL of dry DMF was stirred at 508C overnight under an argon
atmosphere. The mixture was poured to iced water and the pre-
cipitated matter was filtered off, washed with water, and dried. A
crude product obtained by recrystallization from methanol was pu-
rified by column chromatography on silica gel (elution with chloro-
form; Rf =0.65). The fluorescent gelator 9 was obtained by recrys-
tallization from ethyl acetate/methanol in a yield of 0.87 g (48%);
IR (KBr): n˜ =3285 (nNH amide A), 1636 (nC=O amide I), 1542 (dN-H
amide II), 754 cmÀ1 (benzothiazole); 1H NMR (400 Hz, CDCl3, TMS,
258C): d=5.99 (d, J=7.0 Hz, 2H; CH2CONH), 7.01 (d, J=8.3 Hz, 2H;
3-PhH), 7.08 (t, J=7.0 Hz, 2H; 5-PhH), 7.34–7.50 (m, 6H; 4-PhH, 5,6-
benzothiazoleH), 7.92 (d, J=7.7 Hz, 2H; 6-PhH), 8.06 (d, J=8.1 Hz,
2H; 4-benzothiazoleH), 8.50 ppm (d, J=9.6 Hz, 2H; 7-benzothiazo-
leH); elemental analysis calcd (%) for C54H68N4O4S2: C 71.96, H 7.60,
N 6.22; found: 70.82, H 7.62, N 6.24; excitation maximum: 354 nm;
emission maximum: 418 nm (ethyl acetate).
Fluorescent gelator 12: Fluorescent gelator 12 was obtained from
a mixture of 1.76 g (5.0 mmol) of 11-(9H-carbazol-9-yl)undecanoic
acid, 1.92 g (5.0 mmol) of l-leucylaminooctadecane, 0.70 g
(5.5 mmol) of DIPC, 0.61 g (5.0 mmol) of DMAP, and 40 mL of di-
chloromethane by a similar procedure to that described for gelator
11. Yield: 1.26 g (35%); Rf =0.83 (chloroform, alumina); IR (KBr): n˜ =
3300 (nNH amide A), 1635 (nC=O amide I), 1541 (dNÀH amide II),
749, 719 cmÀ1 (carbazole); elemental analysis calcd (%) for
C47H77N3O2: C 78.83, H 10.84, N 5.87; found: C 78.23, H 11.11, N
6.27; excitation maximum: 408 nm; emission maximum: 485 nm
(chloroform).
Fluorescent gelator 10: A mixture of 10.50 g (40 mmol) of cyclo(l-
asparaginyl-l-phenylalanyl),[36] 7.24 g (40 mmol) of 6-bromo-1-hexa-
nol, 11.78 g (40 mmol) of DPTS (4-dimethylamino pyridinium p-tol-
uenesulfonate), 5.55 g (44 mmol) of DIPC (N,N’-diisopropylcarbodii-
mide), and 150 mL of dry DMF was stirred overnight at 358C. After
evaporating DMF, the residue was dissolved in 200 mL of hot 1-
Fluorescent gelator 13: A mixture of 1.66 g (5.0 mmol) of fluores-
cein, 0.56 g (10.0 mmol) of potassium hydroxide, 0.83 g (5.0 mmol)
of potassium iodide, 3.36 g (11.0 mmol) of 1-bromohexadecane,
and 20 mL of dry DMF was stirred at 508C overnight under an
argon atmosphere. An obtained crude product was purified by
column chromatography on alumina (elution with chloroform; Rf =
&
&
Chem. Eur. J. 2016, 22, 1 – 12
www.chemeurj.org
10
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

Full Paper
0.62). The compound 13 was obtained by recrystallization from
hexane/methanol in a yield of 2.43 g (62%). IR (KBr): n˜ =2920, 2851
(nCH2), 1718 (nC=O lactone), 1603, 1286, 1254 cmÀ1; elemental
analysis calcd (%) for C52H76O5: C 79.95, H 9.81; found: C 79.14, H
10.37; excitation maximum: 380 nm; emission maximum: 547 nm
(chloroform).
Acknowledgements
The present research was supported in part by a Grant-in-Aid
for Scientific Research (C) (No. 15K05623) from the Ministry of
Education, Culture, Sports, Science and Technology of Japan.
Compound 14: This compound was prepared from 7.65 g
(20 mmol) of l-isoleucylaminooctadecane, 4.38 g (20 mmol) of n-
dodecanoy chloride, 5.4 mL (40 mmol) of triethylamine, and
300 mL of dry THF by a similar procedure to that described for ge-
lator 3. A crude product was dissolved in hot ethyl acetate and
cooled to room temperature. The formed gel was ground by me-
chanical stirrer and filtered, followed by drying. Yield: 10.17 g
(90%); IR (KBr): n˜ =3288 (nNH amide A), 1634 (nC=O amide I),
1542 cmÀ1 (dNÀH amide II); elemental analysis calcd (%) for
C35H69BrN2O2: C 76.53, H 12.85, N 4.97; found: C 76.44, H 12.65, N
5.12.
Keywords: fluorescence · fluorescence microscopy · gels ·
gelation · gelators
[1] A. V. Lipowitz, Liebigs Ann. 1841, 38, 348.
[2] S. Yamamoto, J. Chem. Soc. Jpn. Ind. Chem. Soc. 1943, 46, 779; Chem.
Abstr. 1952, 46, 7047i.
[3] P. Terech, R. G. Weiss, Chem. Rev. 1997, 97, 3133.
[4] J. H. van Esch, B. L. Feringa, Angew. Chem. Int. Ed. 2000, 39, 2263;
Angew. Chem. 2000, 112, 2351.
[5] L. A. Estroff, A. D. Hamilton, Chem. Rev. 2004, 104, 1201.
[6] P. Dastidar, Chem. Soc. Rev. 2008, 37, 2699.
[7] S. Banerjee, R. K. Das, U. Maitra, J. Mater. Chem. 2009, 19, 6649.
[8] M. Suzuki, K. Hanabusa, Chem. Soc. Rev. 2009, 38, 967.
[9] M. Suzuki, K. Hanabusa, Chem. Soc. Rev. 2010, 39, 455.
[10] J.-L. Li, X.-Y. Liu, Adv. Funct. Mater. 2010, 20, 3196.
[11] G. John, B. V. Shankar, S. R. Jadhav, P. K. Vemula, Langmuir 2010, 26,
17843.
Gelation test
A gelation test was performed by the upside-down testtube
method. A weighed sample and 1 mL of solvent in a septum-
capped test tube with internal diameter of 14 mm was heated
until the solid dissolved. The resulting solution was cooled at 258C
for 2 h and then the gelation was checked visually. When no fluid
ran down the wall of the test tube upon inversion of the test tube,
we judged it to be gel. The gelation ability was estimated by the
minimum gel concentration (MGC), which is the minimum concen-
tration of a gelator necessary for gelation at 258C. The unit is gLÀ1
(gelator per solvent).
[12] H. Svobodovꢃ, V. Noponen, E. Kolehmainen, E. Sievꢁnen, RSC Adv. 2012,
2, 4985.
[13] S. S. Babu, S. Prasanthkumar, A. Ajayaghosh, Angew. Chem. Int. Ed. 2012,
51, 1766; Angew. Chem. 2012, 124, 1800.
[14] A. Y.-Y. Tam, V. W.-W. Yam, Chem. Soc. Rev. 2013, 42, 1540.
[15] J. Raeburn, A. Z. Cardoso, D. J. Adams, Chem. Soc. Rev. 2013, 42, 5143.
[16] G. Yu, X. Yan, C. Han, F. Huang, Chem. Soc. Rev. 2013, 42, 6697.
[17] M. D. Segarra-Maset, V. J. Nebot, J. F. Miravet, B. Escuder, Chem. Soc. Rev.
2013, 42, 7086.
[18] S. S. Babu, V. K. Praveen, A. Ajayaghosh, Chem. Rev. 2014, 114, 1973.
[19] D. K. Kumar, J. W. Steed, Chem. Soc. Rev. 2014, 43, 2080.
[20] V. K. Praveen, C. Ranjith, N. Armaroli, Angew. Chem. Int. Ed. 2014, 53,
365; Angew. Chem. 2014, 126, 373.
[21] Y. Lan, M. G. Corradini, R. G. Weiss, S. R. Raghavanc, M. A. Rogers, Chem.
Soc. Rev. 2015, 44, 6035.
Sample preparation for TEM
[22] C. Geiger, M. Stanescu, L. Chen, D. G. Whitten, Langmuir 1999, 15, 2241.
[23] X. Zhang, R. Lu, J. Jia, X. Liu, P. Xue, D. Xu, H. Zhou, Chem. Commun.
2010, 46, 8419.
[24] P. Rajamalli, E. Prasad, Org. Lett. 2011, 13, 3714.
[25] F. Rodrꢄguez-Llansola, J. F. Miravet, B. Escuder, Chem. Commun. 2011, 47,
4706.
For the TEM samples, fluorescent gelators were dissolved in sol-
vents at the concentration of 2 mgmLÀ1, and a droplet of the solu-
tion was placed on MICA-coated grid (copper, 400 mesh). The sol-
vent was evaporated spontaneously at room temperature for 2 h,
and then dried by a vacuum pump overnight. The samples were
negatively stained with osmium tetroxide vapor (2 wt% acetone
solution) for 10 h.
[26] P. Jana, S. Maity, S. K. Maity, P. K. Ghorai, D. Halder, Soft Matter 2012, 8,
5621.
[27] C. Ou, J. Zhang, X. Zhang, Z. Yang, M. Chen, Chem. Commun. 2013, 49,
1853.
[28] C. Zhao, B. Bai, H. Wang, S. Qu, G. Xiao, T. Tian, M. Li, J. Mol. Struct.
2013, 1037, 130.
Sample preparation for FE-SEM
[29] D. B. Rasale, I. Maity, A. K. Das, Chem. Commun. 2014, 50, 8685.
[30] N. Mizoshita, Y. Suzuki, K. Hanabusa, T. Kato, J. Mater. Chem. 2002, 12,
2197.
[31] K. Kato, S. Hayashi, Develop. Growth Differ. 2008, 50, 381.
[32] M. M. Frigault, J. Lacoste, J. L. Swift, C. M. Brown, J. Cell Sci. 2009, 122,
753.
Fluorescent gelators were dissolved in solvents at the MGC and
a droplet of the hot solution was placed on a sample table. The
solvent was evaporated spontaneously at room temperature for
2 h, and then dried by a vacuum pomp overnight. The sample
table was shadowed to approximately 10 nm thickness with Pt by
a Hitachi ion sputtering apparatus E-1010.
[33] K. Hanabusa, K. Harano, M. Fujisaki, Y. Nomura, M. Suzuki, Macromol.
Symp., unpublished results.
[34] M. Suzuki, S. Owa, M. Kimura, A. Kurose, H. Shirai, K. Hanabusa, Tetrahe-
dron Lett. 2005, 46, 303.
[35] S. Kobayashi, K. Hanabusa, N. Hamasaki, M. Kimura, H. Shirai, S. Shinkai,
Chem. Mater. 2000, 12, 1523.
Sample preparation for FM and CLSM
[36] K. Hanabusa, H. Fukui, M. Suzuki, H. Shirai, Langmuir 2005, 21, 10383.
For the prepared specimens, fluorescent gelators were dissolved in
solvents at the MGC and a few droplets of the hot solution were
placed on a hole-slide glass, and then a cover glass was put on the
hole-slide glass.
Received: July 12, 2016
Published online on && &&, 0000
Chem. Eur. J. 2016, 22, 1 – 12
www.chemeurj.org
11
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
&
These are not the final page numbers! ÞÞ

Full Paper
FULL PAPER
Loading image: A 3D image of a DMSO
gel of a fluorescein-containing gelator
was constructed by confocal laser scan-
ning microscopy. The image was collect-
ed every 0.1 mm to a depth of 9 mm, re-
sulting in a total of 90 images. The ob-
tained 3D image of a 200ꢂ200 mm area
is shown here. The large micrometer-
size aggregates spread out three-dimen-
sionally and the resulting 3D networks
trap solvents in space, with gelation oc-
curring as the final step.
&
Supramolecular Chemistry
K. Hanabusa,* T. Ueda, S. Takata,
M. Suzuki
&& – &&
Synthesis of Fluorescent Gelators and
Direct Observation of Gelation with
a Fluorescence Microscope
&
&
Chem. Eur. J. 2016, 22, 1 – 12
www.chemeurj.org
12
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!