Supramolecular assembly of self-labeled amphicalixarenes

Article abstract of DOI:10.1002/chem.200802008

The synthesis and precise supramolecular organization of new amphicalixarene 4 bearing four rodlike aligned fluorescent terephthalic benzamide moieties in the upper rim is reported. The aggregation of 4 was monitored by a combination of fluorescence, cond

Full text of DOI:10.1002/chem.200802008

FULL PAPER
DOI: 10.1002/chem.200802008
Supramolecular Assembly of Self-ACTHUNTGRNEUNGLabeled Amphicalixarenes
Miriam S. Becherer,[a] Boris Schade,[b] Christoph Bçttcher,*[b] and Andreas Hirsch*[a]
Abstract: The synthesis and precise
supramolecular organization of new
amphicalixarene 4 bearing four rodlike
aligned fluorescent terephthalic benz-
amples of structurally defined micelles
reported previously, this new type of
micelles, the other highly important
finding from this study is the fact that
guest molecules directly influence the
micellar organization because they can
interact with both the free amphiphiles
below the critical micelle concentration
(cmc) or the micellar aggregate itself.
These two types of interactions are es-
pecially pronounced in the case of the
cationic pyrene derivative 10, which
binds electrostatically with the amphi-
phile 4. In addition, a very unique
membrane structure exhibiting a regu-
lar hexagonal pattern of 5 nm pores is
formed by 4 at pH 4.
self-labeled amphiphile serves as
a
fluorescence reporter with the tereph-
thalic units additionally providing an
extended cavity structure in the hydro-
philic part, which facilitates the inclu-
sion of guest molecules. In addition to
the observed formation of well-defined
ACHTUNGTRENNUNGamide moieties in the upper rim is re-
ported. The aggregation of 4 was moni-
tored by a combination of fluorescence,
conductometry, and cryo-TEM meas-
urements at different pH values and re-
vealed significant structural differences.
Most interestingly, we found that exact-
ly 12 molecules of 4 assemble to form a
spherical and structurally persistent mi-
celle at pH 7, which coexists with rod-
like micelles. In contrast to other ex-
Keywords: amphiphiles
· calixar-
enes · fluorescence spectroscopy ·
micelles · scanning probe micros-
copy
Introduction
by employing image-processing and 3D reconstruction tech-
niques and revealed an almost spherical aggregate of 7.5 nm
diameter formed by seven molecules of 1 in a special C2-
symmetrical arrangement. The negatively charged carboxylic
groups are homogeneously distributed over the surface of
the micelle by nonlinear repulsive forces. Thus the T-shape
of 1 induces pronounced backfolding and thereby reduced
flexibility in the aggregate. The dodecyl chains of 1 are di-
rected towards the core of the micelle, which can additional-
ly accommodate hydrophobic guests. This behavior was
proven by steady-state fluorescence experiments with
pyrene, which showed that it is solubilized in the micelles of
1 sensing moderate polarity as concluded from the 11:13
ratio.[2] In subsequent investigations we discovered further
examples of structurally defined and shape-persistent mi-
celles composed of amphiphilic derivatives of C60 (2 and 3)
bearing a [5:1] and [3:3] hexa-addition pattern, respectively.
In both cases it was also possible to determine three-dimen-
sional structural information from the detected assemblies,
with two different ultrastructures being found for 2, namely
rodlike assemblies and spherical micelles, depending on the
pH conditions.[3,4]
Recently, we introduced the T-shaped dendrocalixarene 1,
which consists of two second-generation Newkome-type
dendrons bearing 18 carboxylic termini at the upper rim and
four dodecyl chains at the lower rim spatially aligned by the
calixarene cage. This T-shaped calixarene is able to form
structurally precisely defined micelles, which has allowed a
detailed description of the three-dimensional architecture of
a micelle with molecular precision for the first time.[1] The
aggregate structure was determined from cryo-TEM images
[a] Dipl.-Chem. M. S. Becherer, Prof. Dr. A. Hirsch
Department of Chemistry and Pharmacy
Interdisciplinary Center of Molecular Materials (ICMM)
Friedrich-Alexander-Universitꢀt Erlangen-Nꢁrnberg
Henkestrasse 42, 91054 Erlangen (Germany)
Fax : (+49)9131-852-6864
E-mail: andreas.hirsch@chemie.uni-erlangen.de
[b] Dr. B. Schade, Priv.-Doz. Dr. C. Bçttcher
Research Center for Electron Microscopy
Department of Chemistry and Biochemistry, Freie Universitꢀt Berlin
Fabeckstrasse 36a, 14195 Berlin (Germany)
Fax : (+49)30-838-56589
Herein, we present a new prototype of an amphiphilic cal-
ixarene 4, which is self-labeled with four fluorescent tereph-
thalic acid moieties covalently intercalated between the
E-mail: bottcher@chemie.fu-berlin.de
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author.
Chem. Eur. J. 2009, 15, 1637 – 1648
ꢂ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1637

upper calixarene rim and the dendritic head groups. The
basic idea was to introduce a couple of beneficial features
that can be exploited to allow an even more detailed investi-
gation of the micellization process. First, the chromophoric
benzamide groups provide an internal fluorescence probe,
which guarantees direct spectroscopic observation of micelle
formation. Second, the comparatively enhanced rigidity
combined with the high contrast properties provided by the
aromatic building blocks attached to the head groups was
expected to yield supramolecular assemblies with even more
stability and thus better quality cryo-TEM data, which in
principle are of comparatively low contrast. In addition,
these units provide an extended cavity structure at the
upper rim, which is expected to facilitate the inclusion of
guest molecules.
1638
www.chemeurj.org
ꢂ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2009, 15, 1637 – 1648

Supramolecular Assembly of Amphicalixarenes
FULL PAPER
The chromophoric benzamide absorbs at around lmax
=
tures. The free carboxylic acids of 8 were attached to the
Newkome dendrimers under standard Steglich conditions.
The pure product 9 was produced by reprecipitation in a
yield of 58%. The final step in the synthesis of this new flu-
orescent calixarene 4 was the cleavage of the tert-butyl
esters with formic acid to introduce 12 free carboxylic acid
groups.
330 nm.[5,6] In steady-state fluorescence measurements
benzamide shows a pronounced Stokesꢃ shift and emits in
the region between 470 and 520 nm. As a consequence, the
micellization of 4 is easily detectable without the addition of
extra fluorescent reporters that could interfere with the
exact mode of micellization. In addition to fluorescence
spectroscopy we report on conductometry measurements as
an independent measure of the aggregation behavior and
the determination of the critical micelle concentration (cmc)
of 4 in water. We also report on the complementary use of
pyrene as a noncovalently bound fluorescence probe to de-
termine the micropolarity of micelles of 4 in steady-state
fluorescence measurements. The studies were carried out at
pH 7 and 9 to investigate the putative effects on aggrega-
tion. We report also on the influence of charged pyrene de-
rivatives 10 and 13 (see Scheme 2) on the formation of mi-
celles to gain more information on guest–host interactions.
Transmission electron microscopy (TEM), especially cryo-
TEM, was used to gather direct structural information on
the structure of supramolecular assemblies formed by 4 at
pH values of interest and in the presence of guest molecules
such as pyrene.
1
All new compounds were fully characterized by H and
13C NMR spectroscopy, elemental analysis, and mass spec-
trometry. The free acid 4 is soluble in phosphate-buffered
water at pH 7 or higher, but not in apolar solvents.
The functionalized pyrene 10, which was used in the fluo-
rescence measurements as a fluorescence antenna, was syn-
thesized according to Scheme 2. The hydroxy group of com-
Scheme 2. Synthesis of 1-(4-trimethylammoniobutyl)pyrene bromide (10):
Reagents and conditions: a) CBr4, PPh3, CH2Cl2, 6 min; b) NMe3, THF,
8 h.
Results and Discussion
mercially available 4-(pyren-1-yl)butanol (11) was replaced
by bromide in a nucleophilic substitution reaction.[8] Subse-
quently, the bromide of 12 was
substituted by trimethylamine
in THF. The resulting ammoni-
um salt 10 was purified by rep-
recipitation. This positively
charged pyrene derivative 10,
The synthesis of the new water-soluble dendrocalixarene 4 is
shown in Scheme 1. The precursor calix[4]arene 5 was syn-
thesized according to reported procedures.[7] To couple the
fluorescent units to the upper rim, 5 was allowed to react
with methyl 4-(chlorocarbonyl)benzoate (6) in THF. The
ester groups of 7 were subsequently deprotected with lithi-
um hydroxide in a water/THF mixture at elevated tempera-
pyrene, and 4-(pyren-1-yl)buty-
ric acid (13) were used to inves-
tigate the aggregation ability of
the terephthal calixarene 4.
Characterization of the aggregation of 4: Excited at l=
333 nm, deprotonated 412À, generated by the action of
12 equivalents of NaOH, emits radiation of around 490 nm.
The light emission is caused by twisted intramolecular
charge transfer (TICT).[9] This TICT arises either from elec-
tron transfer between twisted donor–acceptor groups or
from a change in the configuration of the amino nitrogen
from pyramidal to planar in the ICT. Fluorescence spectra
of 412À are shown for a concentration range between 4.5ꢄ
10À8 and 2.7ꢄ10À4 molLÀ1 in Figure 1. The absorption spec-
trum of 412À is displayed in the inset.
The fluorescence spectra show an increase in intensity of
the benzamide band (IBA) on increasing the concentration of
4 until a maximal intensity (f) is reached. IBA strongly de-
creases on further increasing the concentration of 4. A plot
of this nonlinear behavior of IBA as a function of the concen-
tration of 4 shows a fluorescence intensity maximum at 2.2ꢄ
10À5 molLÀ1 of 4 (Figure 2).[10]
Scheme 1. Synthesis of the terephthal calixarene 4. Reagents and condi-
tions: a) i. SOCl2, DMF, 708C, 4 h; ii. THF, À108C to room temperature;
b) LiOH, H2O, THF, 6 h, 608C; c) EDC, HOBt, DMAP, DMF, 08C to
room temperature, 72 h; d) formic acid.
Chem. Eur. J. 2009, 15, 1637 – 1648
ꢂ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
1639

A. Hirsch, C. Bçttcher et al.
Figure 3. Variation of conductivity versus the concentration of 412À for
the determination of the cmc in pure water (k=2 mS, Tref =258C).
Figure 1. Intensity of the benzamide band IBA of 412À in water at low
(a) 4.5ꢄ10À8, b) 9.1ꢄ10À7, c) 3.9ꢄ10À6, d) 8.2ꢄ10À6, e) 1.7ꢄ10À5, f) 3.5ꢄ
10À5 molLÀ1 (gray lines)) and high (g) 5.1ꢄ10À5, h) 8.7ꢄ10À5, i) 1.5ꢄ10À4
,
k) 2.7ꢄ10À4 molLÀ1 (black lines)) concentrations of
4 (lex =333 nm;
Accordingly, the amphicalixarene 4 was investigated with-
out auxiliary dyes in buffered water at pH 7 (Na/K phos-
phate) and 9 (Na borate), respectively. The benzamide emis-
sion band experiences a shift to 485 nm at pH 7 and to
496 nm at pH 9, respectively, which demonstrates its sensi-
inset: absorption spectrum of 4).
tivity towards the solvent environment.[9] The plots of IBA
/
IBA,max as a function of the concentration of 4 (Figure 4)
follow qualitatively the same trends as those determined for
4 treated with 12 equivalents of NaOH.
Figure 2. Intensity of the benzamide band IBA of 412À at 485 nm versus the
concentration of 412À in pure water (k=2 mS, lex =333 nm).
The initial increase in IBA is due to the steady increase in
the calixarene concentration. Beyond the maximum the
fluorescence energy is quenched by energy transfer between
the benzamide moieties, which come into close proximity on
micelle formation. Hence, we associate the maximum of the
fluorescence with the cmc of amphicalixarene 4 (cmc0), that
is, 2.2ꢄ10À5 molLÀ1.
Figure 4. IBA/IBA,max of 4 versus the concentration of 4 at pH 7 (&) and 9
&
( ).
To support this result the cmc0 was also determined by
conductivity measurements. The reliability of this method
was initially proven by determination of the known cmc of
sodium dodecyl sulfate (9.0ꢄ10À3 molLÀ1), which is in good
accord with the literature value within the limits of experi-
mental error (8.1ꢄ10À3 molLÀ1).[11] The conductivity of the
calixarene 412À solution increased linearly with concentration
in two sections, and can be fitted by two straight lines
(Figure 3). Their intersection at a concentration of 2.3ꢄ
10À5 molLÀ1 coincides within experimental error with the
value of cmc0 determined by fluorescence spectroscopy.
Low fluorescence intensities for the benzamide moieties
are observed at both pH values at high concentrations of 4,
which indicates a close spatial association of the benzamide
units within the aggregates. At pH 7, the cmc of 4 (cmc(4))
is 5.0ꢄ10À5 molLÀ1 and a value of 3.7ꢄ10À5 molLÀ1 deter-
mined at pH 9. The smaller cmc(4) value at pH 9 compared
with neutral conditions indicates a modified interaction be-
tween the chromophores and hence a different structural or-
ganization. Electron microscopy studies indeed showed dif-
ferent aggregation behavior depending on the pH (see
below). Both cmc values are higher than in pure water
1640
www.chemeurj.org
ꢂ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2009, 15, 1637 – 1648

Supramolecular Assembly of Amphicalixarenes
FULL PAPER
(Table 1). Hence, the different buffer salts and the resulting
change in ionic strength, as well as different pH values,
modify the aggregation behavior of calixarene 4.
Table 1. Critical micelle concentrations (cmcs) of 4 at pH 7 and 9 and
the intersection points of the linear analysis of the I1 intensities of
pyrene, 10, and 13.
cmc of 4 [molLÀ1
]
Intersection points [mol LÀ1] of
the linear fitted slopes of I1/I0
for the pyrene moiety of
pyrene, 10, and 13 versus [4][a]
cmc0
2.3ꢄ10À5
pH 7.0
pH 9.0
pH 7.0
pH 9.0
cmc(4)
5.6ꢄ10À5
4.1ꢄ10À5
7.3ꢄ10À5
4.0ꢄ10À5
6.3ꢄ10À5
1.0ꢄ10À4
3.7ꢄ10À5
2.1ꢄ10À6
5.2ꢄ10À5
4.3ꢄ10À5
–
cmc
cmc
cmc
N
8.0ꢄ10À6
6.0ꢄ10À6
4.2ꢄ10À7
3.0ꢄ10À7
1.9ꢄ10À5
–
–
2.4ꢄ10À5
–
–
Figure 6. Typical cryo-TEM images of 4 in buffered water at different pH
values and in the presence of pyrene. Pure 4 forms predominantly rod-
like and spherical micellar aggregates at pH 7 (a) and only almost uni-
form spherical micelles at pH 9 (b). After the addition of pyrene the di-
mensions of the aggregates at pH 7 remain unaffected (c), whereas at
basic pH the loaded micelles are less uniform providing a larger particle
size distribution (d). The formation of micellar end-caps can be detected
(see arrows in a and c) irrespective of the addition of pyrene.
cmc(10)
cmc(13)
–
[a] See ref. [12].
Structural characterization by electron microscopy: As has
been extensively shown in previous publications[1,3,4] cryo-
TEM provides direct and detailed evidence of the aggregate
formation of amphiphilic dendrimers. However, the low con-
trast associated with this technique is a big problem and re-
quires the additional application of image-averaging tech-
niques or the use of contrasting materials. In this work, the
introduction of benzamide entities boosts the contrast and
allows a better visualization of the aggregates without fur-
ther treatment of the data (Figure 5).
al feature of double-layered amphiphilic architectures. The
rodlike micelles have a diameter of around 7.1 nm, which is
only slightly larger than twice the length of the fully stretch-
ed molecule (3.2 nm), which also hints at the double-layer
arrangement of the amphiphiles. In contrast, the spherical
micelles are slightly larger with a diameter of around 8.4 nm
(a volume that allows 12 molecules of 4 to be accommodat-
ed, as discussed below). We conclude that the molecular
packing is slightly different in both of these aggregates,
which allows two types of curvature. This observation is
very similar to our earlier examinations of dendrofullerene
2,[3] which forms rodlike and spherical micelles depending
on the pH of the solutions.
Calixarene 4 forms solely spherical micelles at pH 9 (Na
borate buffer), which, with an average diameter of only
6.3 nm, are clearly smaller than those at neutral pH (Fig-
ure 5b). From this data, it can be concluded that denser
packing of the micelles occurs under basic conditions in
comparison with the aggregates obtained at neutral pH. The
resulting larger curvature may arise from greater bending of
the dendrons as a result of mutual repulsion of the fully
charged carboxylic groups at this pH value.
Figure 5. The introduction of aromatic terephthalamides into 4 causes
visibly enhanced contrast in the cryo-TEM images of micellar assemblies
(a) compared with those of dendrocalixarene 1 without the aromatic
spacer (b).
In comparison with our earlier investigations,[1,3,4] calixar-
ene 4 exhibits slightly different aggregation behavior as we
have evidently detected conditions (neutral pH) under
which two different aggregation states are in equilibrium
(compounds 1 and 2 show uniform entities exclusively at
each pH). This observation coincides with the behavior
found for classical detergents in which spheres and rods are
occasionally found in equilibrium depending on the concen-
tration,[13a] pH, and ionic strength.[13b] A typical structural
feature, which indicates the different spatial demand of the
All samples used for TEM had a concentration between
7.5ꢄ10À4 and 10.4ꢄ10À4 molLÀ1, which is clearly above the
value of cmc(4). The aggregates visualized by cryo-TEM
showed different structures depending on the pH. At pH 7
(Na/K phosphate-buffered), rodlike and spherical micelles
were observed to coexist (Figure 6a). Both types of aggre-
gates exhibit low density areas in their central part, which
can be attributed to the hydrophobic core, a typical structur-
Chem. Eur. J. 2009, 15, 1637 – 1648
ꢂ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
1641

A. Hirsch, C. Bçttcher et al.
molecules in spheres and rods, are the swollen end-caps.
They can also be found in the rods of 4 (see arrows in Fig-
ure 6a). The slightly increased diameter of the caps coin-
cides well with the diameter of the spheres.
The 3D density map obtained shows the accommodation
of 12 molecules in a perfectly D2-symmetrical manner. This
structure differs from that obtained from the structurally
similar compound 1. The packing of the head groups of 4 is
denser in the aggregates, most probably due to the smaller
head-group volume (first-generation dendrons in 4 versus
second-generation dendrons in 1). Figure 8 illustrates these
differences.
In addition, all the spherical aggregates of 4 display an ul-
trastructural organization similar to those of the structurally
persistent micelles obtained from calixarene 1 or the den-
drofullerenes 2 and 3. A gallery of class averages proving
this finding is provided in Figure 7a for the spherical mi-
celles formed at pH 7. Each of the so-called class sum
images in the gallery represents a different spatial orienta-
tion of the micelle. Taking these images together a complete
three-dimensional structure determination of the aggregate
can be achieved, as described previously.[1] Figure 7b shows
the surface representation of the reconstruction based on a
data set of 11041 images of individual aggregates.[14]
Figure 8. Spatial conformations of the head groups of 4 (left) and 1
(right) found in structurally persistent spherical micelles. The smaller
space required by 4 allows denser packing of the second-generation den-
dritic head groups compared with the third-generation head groups in
compound 1.
Whereas the second-generation dendrons of 1 protrude
widely in different directions (Figure 8, right), the less volu-
minous first-generation dendrons of 4 are packed more
tightly (Figure 8, left), which allows a larger aggregation
number of 12 compared with seven in the case of 1.
A unique supramolecular architecture at pH 4: The results
of the TEM analysis of 4 at pH 4 were both very interesting
and surprising. As protonation of the head groups drastically
reduces its solubility, slow precipitation is observed upon
acidification. Although no ordered structural features were
expected from the precipitate we studied the cloudy suspen-
sion by cryo-TEM. To our surprise very large and wrinkled
membrane sheets of micrometer size appeared, which
showed a regular pattern of pores with a uniform diameter
of 5.0 nm. In those areas where the sheets were unwrinkled
the hexagonal-ordered pattern of the pores became appar-
ent (Figure 9).
The tilt series (À22.5 to 22.58 in 58 steps) of these
cobweb-like aggregates reveal a membrane thickness of
6.0 nm if the sheets are orientated in a side-view orientation,
for example, with its edges parallel to the electron beam
(Figure 10, right).
Figure 7. a) Selected reference free class sum images of 11041 individual
aggregates gathered from electron micrographs of a cryo-negative stain
(phosphotungstic acid stain) sample of 4 in buffered water at pH 7. The
class sum images represent different spatial orientations of the three-di-
mensional micelles and show different density patterns and dimensions
due to its inherent structure. b) Upper row: Transparent surface represen-
tation of the 3D density map calculated from 11041 images of individual
micelles formed by 4 at pH 7.2. The reconstruction is shown in the direc-
tion of the inherent two-fold symmetry (D2) axes. Lower row: Fitting of
the molecular models of 4 to the 3D density map shown above. Twelve
individual molecules of 4 can be very accurately accommodated in the re-
constructed volume.
One can therefore conclude that the network itself con-
sists of interconnected cylindrical components, with a cross-
section diameter of 6.0 nm, that undergo regular bifurca-
tions. The cylinder diameter of 6.0 nm coincides well with
the molecular double-layer dimension.
Similar porous membranes have recently been described
by Kim et al.[15] for dumb-bell-shaped amphiphiles forming a
regular hexagonal pattern of 25 nm pores, which can be
thermoreversibly switched to form closed vesicles featuring
a continuous membrane layer.
1642
www.chemeurj.org
ꢂ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2009, 15, 1637 – 1648

Supramolecular Assembly of Amphicalixarenes
FULL PAPER
Transport capacities of the amphicalixarene 4 aggregates:
To examine the transport capacities and the possible effects
of additives on the aggregation process, pyrene and its sub-
stituted derivatives 10 and 13 were used as guest molecules
and probes in the presence of terephthal calixarene 4. By
utilizing these antennae, fluorescence experiments were per-
formed at pH 7 and 9 at a constant concentration of 6.0ꢄ
10À7 molLÀ1 of the antennae.
Transport of pyrene: Upon excitation at 333 nm, in addition
to the broad emission band of 4, characteristic and well-de-
fined vibronic emission bands of the pyrene moiety appear
in the spectra (Figure 11). At pH 7 and 9 the benzamide
bands are located at 485 nm and 495 nm, respectively.
IBA increases on increasing the concentration of 4 and
reaches a maximum at 4.1ꢄ10À5 molLÀ1 (pH 7), which is at-
tributed to the cmc of 4 in the presence of pyrene (cmcACHTUNGTRENNUNG(4py))
(Figure 12).[10] This value is slightly smaller than cmc(4).
Beyond this point IBA decreases again due to enforced intra-
and intermolecular interactions within the micelles and
eventually tends to zero. At pH 9 the benzamide bands of 4
show a slightly different behavior. IBA rises up to a concen-
tration of 2.1ꢄ10À6 molLÀ1, features a local minimum at
1.5ꢄ10À5 molLÀ1, and reaches a second maximum at 4.1ꢄ
10À5 molLÀ1. This means that 4 starts to form aggregates at
a concentration of 2.1ꢄ10À6 molLÀ1. At higher concentra-
tions the fluorescence intensity is increasingly quenched due
to the close contact of the benzamide moieties. At the next
maximum (4.1ꢄ10À5 molLÀ1), a second cmc is obtained,
which corresponds to the value of cmc(4) obtained at pH 7.
As the monomer emission of pyrene is highly sensitive to
the polarity of the environment the intensity ratio between
the first and third vibronic bands I1 and I3 can be used to
monitor the polarity of the surrounding medium. In water
this ratio is about 1.9 and in apolar solvents, like dodecane
or hexane, it lies in the range 0.5–0.6.[16] In Figure 13 the
fluorescence intensity ratios of pyrene (I1/I3) at pH 7 and 9
are plotted as a function of the concentration of 4.
At low concentrations of 4 pyrene is predominantly locat-
ed in the aqueous phase both at pH 7 and 9, as determined
by the high I1/I3 ratio (1.9). At pH 7 the value of the I1/I3
ratio starts to decrease at around 8.0ꢄ10À6 molLÀ1 of 4 and
reaches a nearly constant value of 0.9 at 4.1ꢄ10À5 m, which
is the value of cmcACTHNUTRGNEUNG(4py). At pH 9 the cmc of the micelles is
Figure 9. a) Cryo-TEM image and b) negatively stained (phosphotungstic
acid) preparation of 4 at pH 4. A porous membrane is formed that shows
pores of 5.0 nm diameter in a hexagonal arrangement, as shown by the
magnified image and its corresponding Fourier transform in (c).
indicated by a slight decrease in the I1/I3 ratio from 1.9 to
1.8. At both pH values the I1/I3 ratios suggest that the
pyrene environment is more polar than a purely aliphatic
environment (I1/I3 =0.5–0.6). Thus, it can be concluded that
pyrene is predominantly exposed to the more polar regions
at the upper rim of the amphicalixarenes in their micellar
aggregates and a pyrene alkyl chain interaction can be ne-
glected. This observation is in accord with previous inclusion
experiments with calixarene 1 and pyrene.[2]
An exponential decrease in the intensity is observed in
the plots of the fluorescence intensities of the first vibronic
band of pyrene (I1) at pH 7 and 9 as a function of the con-
centration of 4 (see the Supporting Information). At the
With these two remarkable examples of a new supra-
molecular architecture formed by very different molecules,
the direction of future investigations is clear; the aim will be
to show to what extent the ultrastructural organization of
porous membranes is dependent on the molecular structure
employed. It would be, for example, very interesting to in-
vestigate the potential of generating membranes with a
tailor-made pore size.
Chem. Eur. J. 2009, 15, 1637 – 1648
ꢂ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
1643

A. Hirsch, C. Bçttcher et al.
pyrene.[18] Both the low and
high concentration sections of
these plots can be fitted by
straight lines.[17] The intersec-
tion points of these lines are
given in Table 1.
No excimer emission due to
the formation of pyrene dimers
is observed, which would be ex-
pected
at
around
lmax =
480 nm.[5] Hence, under these
conditions no aggregation of
pyrene takes places within the
micelles of 4 at either pH 7 or
9.
Figure 10. Cryo-TEM images showing in detail the porous membrane formed by 4 at pH 4 (cf. Figure 9) at dif-
ferent tilt angles (À22,5, 0, and +22,58 from left to right) revealing the membrane layer thickness of 6 nm
(right) (bar: 50 nm).
Figure 11. Fluorescence spectra of 4 in the presence of pyrene (6.10ꢄ
10À7 molLÀ1) at varying concentrations of 4 (a) 1.9ꢄ10À6, b) 4.8ꢄ10À6
,
c) 7.2ꢄ10À6 (dashed lines), d) 8.2ꢄ10À6, e) 3.1ꢄ10À5, f) 5.3ꢄ10À5 molLÀ1
(black lines)) and of 6.0ꢄ10À7 molLÀ1 pyrene (gray line) in buffered solu-
tion at pH 7 (lex =333 nm).
Figure 13. I1/I3 of pyrene versus the concentration of 4 at pH 7 (&) and 9
&
( ) in the presence of pyrene (6.10ꢄ10À7 molLÀ1).
It is possible that the structural effects of the aggregation
of 4 at different pH values and a probable influence of guest
molecules on the supramolecular organization of the host
nanocontainers may be deduced from these detailed data.
Cryo-TEM investigations should indicate if the addition of
pyrene results in changes in the supramolecular organization
of the micelles. Again the micrographs of the mixtures show
rods and spherical micelles at pH 7 and only spherical mi-
celles at pH 9 (Figure 6c). The average diameters of 8.2 nm
for spherical micelles and 6.8 nm for rod-shaped aggregates
coincide with those aggregate dimensions found for the un-
loaded micelles at pH 7, which suggests that these aggre-
gates are not distorted upon the uptake of guest molecules.
Swollen end-caps can also be detected, as in the unloaded
micelles (see the arrows in Figure 6c).
Different behaviour was observed under basic conditions
(Figure 6d). The loaded micelles are less uniform compared
with the unloaded aggregates at the same pH. Although the
average diameter of 6.8 nm is only slightly larger than the
6.3 nm for the unloaded micelles, a notable number of ag-
gregates have diameters of the same magnitude as the
spherical micelles at neutral pH (8.2 nm). As pointed out
above, pyrene must be involved in a pyrene–dendron associ-
Figure 12. Intensity of the benzamide band IBA of 4 versus the concentra-
tion of 4 in the presence of pyrene (6.10ꢄ10À7 molLÀ1) at pH 7 (&) and 9
&
( ).
lowest concentration of 4 (2.8ꢄ10À8 molLÀ1) I1 is nearly
equal to the I1 intensity of pyrene in the absence of 4 at
pH 7 and 9. The same quenching behavior was found for 1:1
complexes of poly(propyleneimine) dendrimers with
1644
www.chemeurj.org
ꢂ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2009, 15, 1637 – 1648

Supramolecular Assembly of Amphicalixarenes
FULL PAPER
ation at the upper rim of 4. Hence, interactions of the guest
molecule with the dendrons might prevent them from bend-
ing as much as the unloaded micelles at basic pH. At this
point it cannot be determined whether only the larger mi-
celles are loaded with pyrene molecules or if different ag-
gregates form as a result of the additive. Note that differen-
ces in diameters are also evident if the three-dimensional ul-
trastructure of structural-persistent micelles is contemplated.
Depending on the spatial orientation of the object, the pat-
tern of the projection image (TEM delivers projection
images) is different and therefore small and large diameters
can be seen.
cmc0). In the presence of pyrene and negatively charged 13
the cmcs tend to be higher than cmc0 with the exception of
cmcACTHNUTRGENNGU(4py) at pH 9. Significantly, cmcAHCUTNGTRNE(NUNG 410) is markedly higher
than cmc(4). The positive charge on 10 seems to disfavor
micelle formation, probably due to the formation of coulom-
bic complexes between 4 and 10 at low concentrations of 4.
This electrostatic association has to be overcome or at least
considerably disturbed during micelle formation in the pres-
ence of 10. Of course, such electrostatically bound com-
plexes are not expected to form with pyrene and 13. The
pronounced attractive interaction between the oppositely
charged molecules 4 and 10 becomes especially apparent if
one looks at the decay of the pyrene emission I1 as a func-
tion of the concentration of 4 (Figure 15). Its decay, which is
These observations clearly show that pyrene, which is fre-
quently used as a fluorescence label to determine the cmcs
of amphiphiles, is not an innocent spectator, but plays an
active role in the micellization process.
Transport of pyrene derivatives: To investigate this issue fur-
ther we also studied the behavior of the pyrene derivatives
10 and 13 as guest molecules. At pH 7 and 9 these molecules
carry one positive or one negative charge, respectively. Be-
cause these pyrene derivatives are themselves amphiphilic
we first had to determine their cmcs and make sure that
they were employed at concentrations below their cmcs in
the subsequent host–guest investigations with 4.[19] The cmc
of 10 at pH 7 is 6.3ꢄ10À5 molLÀ1 and that of 13 is 1.0ꢄ
10À4 molLÀ1. Thus we prepared stock solutions of 10 and 13
with a concentration of 6.0ꢄ10À6 molLÀ1 to ensure that they
do not form micelles on their own. Fluorescence emission
spectra (lex =333 nm) show the expected vibronic structure
characteristic of the pyrene chromophore between 374 and
445 nm as well as the expected benzamide emission of am-
phicalixarene 4 at around 490 nm. The cmcs of 4 in the pres-
Figure 15. Intensity ratio I1/I0 of the pyrene-based emissions of 10 and 13
as a function of the concentration of 4 in the presence of 10 (6.10ꢄ
10À7 molLÀ1) at pH 7 ( ) and 9 ( ) and in the presence of 13 (6.10ꢄ
*
*
10À7 molLÀ1) at pH 7 (&) and 9 ( ).
&
ence of 10 and 13 (cmcACTHNUTRGENN(UG 410) and cmcAHCUTNGTRNEN(UNG 413)) were obtained
from plots of IBA/IBA,max as a function of the concentration of
4 (Figure 14). The maximum values of the plots represent
the corresponding cmcs (Table 1).
In all cases the cmcs are slightly different to those ob-
tained for 4 in the absence of the guest molecules (cmc(4),
due to electronic communication between the two chromo-
phores located in close proximity, proceeds almost instanta-
neously compared with the decay of 13, even well below the
cmcACHTUNRTGNEUN(G 410). This provides very strong corroboration for the
suggestion that electrostatic coulombic complexes are
formed between 4 and 10 prior to the micelle formation of
4. Nevertheless no pyrene excimers were observed during
the fluorescence experiments. This shows that the pyrene
moieties are separated from each other. Hence, they are
most probably located within the calixarene cavity. A linear
curve analysis of the I1 progression at high and low concen-
trations of calixarene 4 in the presence of 10 and 13 reveals
intersection points for each pair of linear plots. The intersec-
tion points for 10 are at 4.2ꢄ10À7 molLÀ1 at pH 7 and 3.0ꢄ
10À7 molLÀ1 at pH 9 (Table 1).
Conclusion
The structurally well-defined micelles of the new amphica-
lixarene 4 are the first to be studied in detail by a combina-
tion of fluorescence, conductometry, and cryo-TEM meas-
Figure 14. Intensities of the benzamide band IBA of 4 versus the concen-
tration of 4 in the presence of 10 (6.10ꢄ10À6 molLÀ1) at pH 7 ( ) and 9
*
&
*
(
) and in the presence of 13 (6.10ꢄ10À7 molLÀ1) at pH 7 (&) and 9 ( ).
Chem. Eur. J. 2009, 15, 1637 – 1648
ꢂ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
1645

A. Hirsch, C. Bçttcher et al.
Sample preparation: All fluorescence samples for cmc determination
were prepared in buffered solution made of ultrapure water from Fluka.
The pH 7 Na/K buffer solution was prepared according to literature pro-
cedures.[22] The borate buffer solution was commercially available from
Fluka. The concentration of pyrene and its derivatives was 6.0ꢄ
10À7 molLÀ1 in all sample solutions. To prepare the pyrene derivative
stock solution a weighed amount was dissolved in acetone. A known
amount of the pyrene/acetone solution was added to a 25 mL flask to
adjust the concentration of pyrene to 6.0ꢄ10À7 molLÀ1 and then the ace-
tone was removed with gaseous nitrogen for 1 h. Then the flask was refil-
led with buffered water. To obtain a sample solution an amount of the
pyrene/acetone stock solution was placed in a flask and the acetone
blown off with nitrogen for 1 h. Then the sample dissolved in buffer was
added and the mixture allowed to equilibrate for 1 h. With these freshly
prepared solutions fluorescence measurements were performed with an
excitation wavelength of 330 or 333 nm. The excitation and emission slit
opening was 3 or 5 nm. The response time was 0.1 s and the scan time
was 0.2 nm for all measurements. For the conductivity measurements, cal-
ixarene 412À was completely deprotonated by dissolving the calixarene in
water and adding 12 equivalents of sodium hydroxide.
urements. In contrast to other examples of structurally de-
fined micelles,[1,3,4] this general approach was facilitated by
the presence of terephthalic acid moieties in the calixarene,
which serve as fluorescence reporter units in this self-labeled
amphiphile. One of the more important findings from this
study is the fact that guest molecules directly influence the
micellar organization because they can interact with both
the free amphiphiles below the critical micelle concentration
(cmc) or with the micellar aggregate itself. These two types
of interactions are especially pronounced in the case of the
cationic pyrene derivative 10, which undergoes electrostatic
binding with the amphiphile 4. Consequently, care has to be
taken if micelle formation is investigated with the help of
only noncovalently bound fluorescence labels. Such guest
molecules are not only spectators, but also play an active
role in micelle formation. Cryo-TEM provides the direct
visual evidence for a fundamental understanding of the
spectroscopic data and reveals that the micellar structures
formed by 4 are predominantly rod-shaped at pH 7 and
solely spherical at pH 9. At the same time we found that ex-
actly 12 molecules of 4 self-assemble to form a spherical and
structurally persistent micelle at pH 7. The addition of
pyrene did not influence the overall appearance of the ag-
gregates at neutral pH. In contrast, at pH 9 the globular mi-
celles are less uniform and most probably reflect the spatial
demand of the guest molecules incorporated into the tightly
packed micellar aggregates. Finally, at pH 4 an intriguing
new kind of supramolecular architecture is observed. A
membrane with a very uniform pattern of 5 nm pores ex-
pands the family of self-assembled structures, which is a sub-
ject of continuing research.
Samples for electron microscopy were prepared from 2 mgmLÀ1 (7.5ꢄ
10À4 molLÀ1) solutions of 4 in standard phosphate (pH 7) or borate
buffer (pH 9). The samples of the loaded micelles were prepared as de-
scribed for the fluorescence investigations with 2.65 mgmLÀ1 (10.0ꢄ
10À4 molLÀ1, pH 7) and 2.76 mgmLÀ1 (10.4ꢄ10À4 molLÀ1, pH 9) solutions
of 4.
Electron cryomicroscopy: Droplets of the sample (5 mL) were applied to
perforated (1 mm hole diameter) 200 mesh grids covered with a carbon
film (R1/4 batch of Quantifoil, MicroTools GmbH, Jena, Germany),
which had been hydrophilized before use by 60 s plasma treatment at
8 W in a Baltec Med 020 device. The supernatant fluid was removed with
filter paper until an ultrathin layer of the sample solution was obtained
spanning the holes of the carbon film. The samples were immediately vit-
rified by propelling the grids into liquid ethane at its freezing point
(90 K) with a guillotine-like plunging device. The vitrified samples were
subsequently transferred under liquid nitrogen into a Tecnai F20 trans-
mission electron microscope (FEI, Oregon, USA) using the Gatan
(Gatan Inc., California, USA) cryoholder and cryostage (Model 626).
Microscopy was carried out at a sample temperature of 94 K by using the
microscopeꢃs low dose routine. Images were recorded with a FEI Eagle
2k CCD camera at an accelerating voltage of 160 kV (binning: 1ꢄ; inte-
gration time: 1 s) at calibrated primary magnifications of 100000(ꢄ) and
62000ꢄ.
Experimental Section
All chemicals and solvents were purchased from Sigma-Aldrich and
Acros Organics or were prepared according to literature procedures.
Dendrimers were synthesized as described previously.[20] tert-Butylca-
lix[4]arene and 5,11,17,23-tetraamino-25,26,27,28-tetrakis(dodecyloxy)ca-
lix[4]arene were synthesized according to the methods of Gutsche and
Iqbal.[21] All reactions were performed by using standard glassware. Dry
solvents were prepared according to literature procedures. Reactions
were monitored by thin-layer chromatography (TLC) using Riedel
de Haꢅn silica gel 60 F 254 aluminium foils and a UV lamp. The products
were purified by flash chromatography (FC) on silica gel 60 (parcel size
0.04–0.063 nm) from Merck. 1H and 13C NMR spectra were recorded with
Bruker Avance 300, JEOL JNM EX 400, JEOL JNM GX 400, and
JEOL A 400 spectrometers. Solvent peaks were used as references. Reso-
nance multiplicities are referred to as s (singlet), d (doublet), t (triplet),
m (multiplet), and unresolved signals as br (broad). Mass Spectra were
measured by using a Micromass Lab Spec (FAB) and a Finnigan MAT
900 spectrometer with 3-nitrobenzyl alcohol as the matrix. MALDI-TOF
mass spectra were acquired on an AXIMA-CFR plus instrument (Kratos
Analytical, Manchester, UK) with cinnamic acid as the matrix. UV/Vis
spectrophotometry was performed on a Shimadzu UV-3102 spectropho-
tometer. Elemental analyses were performed by combustion and gas
chromatography with an EA 1110 CHNS analyzer (CE Instruments).
Fluorescence spectroscopy was performed on a Shimadzu RF-5301 PC
Cryonegative staining electron microscopy: Droplets of the sample
(5 mL) were applied to 200 mesh copper grids covered with a carbon film,
which had been hydrophilized before use by 60 s plasma treatment at
8 W in a Baltec Med 020 device. After 30 s the supernatant fluid was re-
moved with filter paper. Subsequently, a droplet of staining solution
(5 mL, phosphotungstic acid at pH 7) was applied for 45 s, blotted with
filter paper, and the sample subsequently frozen in liquid ethane (see
above). The vitrified samples were subsequently transferred under liquid
nitrogen into a Philips CM12 transmission electron microscope (FEI,
Oregon, USA) using the Gatan (Gatan Inc., California, USA) cryoholder
and cryostage (Model 626). Microscopy was carried out at a sample tem-
perature of 94 K using the low-dose protocol of the microscope at a pri-
mary magnification of 58300(ꢄ) and 300(ꢄ) and an accelerating voltage
of 100 kV (LaB6 illumination). All image processing and 3D reconstruc-
tion calculations of the image data were performed by using the Imagic 5
software (Image Science GmbH, Berlin, Germany), as described else-
where.[1,3,4]
Synthesis of 5,11,17,23-tetraamino-25,26,27,28-tetrakis(dodecyloxy)ca-
lix[4]arene (5): 5,11,17,23-Tetranitro-25,26,27,28-tetrakis(dodecyloxy)ca-
lix[4]arene (3.76 g, 2.95 mmol) was dissolved in isopropanol (300 mL).
Pd/C (379 g, 10%) and hydrazine hydrate (7.17 mL, 148 mmol) were
added to this solution. The reaction mixture was stirred at reflux for 48 h.
After cooling to 408C it was filtered through Celite and the residue
washed with CHCl3. The product was obtained after precipitation with
Fluorescence spectrophotometer. Conductivity was measured with
Cond 340i handheld conductivity meter from WTW.
a
1646
www.chemeurj.org
ꢂ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2009, 15, 1637 – 1648

Supramolecular Assembly of Amphicalixarenes
FULL PAPER
CHCl3/MeOH in a yield of 86% as a light yellow powder (3.24 g,
2.54 mmol).
added and the resulting solution stirred for 72 h. Then the solvent was re-
moved and the crude dissolved in CHCl3 (50 mL) and washed with citric
acid (10%, 50 mL) and brine (50 mL). Reprecipitation with CHCl3/
MeOH furnished the product as a yellow powder in a yield of 58%
(961.1 mg, 0.29 mmol).
1H NMR (CDCl3, 400 MHz, RT): d=0.87 (m, 12H; CH3), 1.25 (m, 72H;
CH2), 1.39 (s, 108H; CH3, tBu), 1.88 (m, 8H; CH2), 2.13 (m, 24H; CH2),
2.20 (m, 24H; CH2), 3.17 (d, 2J=13.8z, 4H; CH2), 3.86 (t, 3J=7.5 Hz,
8H; CH2), 4.46 (d, 2J=13.6 Hz, 4H; CH2), 7.05 (s, 8H; CHarom), 7.34 (s,
4H; NH), 7.82 (m, 8H; CHarom, benzyl), 7.65 (m, 8H; CHarom, benzyl),
8.14 (s, 4H; NH-CO); 13C NMR (CDCl3, 100.5 MHz, RT): d=14.0
(CH3), 22.6, 26.3 (CH2), 28.0 (CH3, tBu), 29.4, 29.7, 29.76, 29.79, 29.9,
29.93, 30.1 (CH2), 30.2, (CH2-Ar), 31.9 (CH2), 58.2 (Cq-NH), 75.4 (CH2-
3
1H NMR (CDCl3, 400 MHz, RT): d=0.84 (t, J=6.7 Hz, 12H; CH3), 1.23
(m, 72H; CH2), 1.81 (m, 8H; CH2), 2.87 (d, 2J=13.2 Hz, 4H; CH2), 3.71
(t, 3J=7.4 Hz, 8H; CH2), 4.26 (d, 2J=12.0 Hz, 4H; CH2), 6.02 ppm (s,
8H; CHarom); 13C NMR (CDCl3, 100.5 MHz, RT): d=14.1 (CH3), 22.6,
25.0, 25.3, 26.4, 29.4, 29.7, 30.0, 30.2 (CH2), 31.1 (CH2-Ar), 31.9 (CH2),
75.1 (CH2-O), 115.7 (CHarom), 135.6 (Carom-CH2), 140.2 (Carom-NH),
150.0 ppm (Carom-O); MS (FAB, NBA): m/z: 1158 [M+H]+.
Synthesis of 5,11,17,23-tetrakis(4-methoxycarbonylphenylcarbonylami-
no)-25,26,27,28-tetrakis(dodecyloxy)calix[4]arene (7)
Methyl 4-(chlorocarbonyl)benzoate (6): Monomethyl terephthalate
(929.0 mg, 5.16 mmol) was dissolved in thionyl chloride (16.3 mL,
137.0 mmol) and a drop of DMF was added. The mixture was stirred at
708C for 4 h. The solvent was then removed and the product immediately
used in the next reaction.
O), 80.7 (CH3, tBu), 120.7 (CHarom), 127.1 (CHarom
, benzyl), 127.4
(CHarom, benzyl), 131.9 (Carom-NHCO), 135.6 (Carom-CO), 136.7 (Carom
-
CH2), 138.1 (Carom-CONH), 154.1 (Carom-O), 164.8, 165.9, 166.4 ppm
(CO); MS (FAB, NBA): m/z: 3640 [M+NaÀ2H]+; elemental analysis
calcd (%) for C196.17H296N8O36·0.17CDCl3 (1833.41): C 70.10, H 8.89, N
3.33; found: C 69.78, H 8.81, N 3.51.
Amide formation: Methyl 4-(chlorocarbonyl)benzoate
6
(1.02 g,
5.1 mmol) was dissolved in THF (10 mL), triethylamine (1.43 mL,
10.32 mmol) added, and the mixture cooled to À108C. 5,11,17,23-Tetra-
Synthesis
[tris(carboxyethyl)methylaminocarbonylphenylcarbonylamino]-
25,26,27,28-tetrakis(dodecyloxy)calix[4]arene (4): Compound
of
Newkome-type
[G-2-OH]
5,11,17,23-tetrakis-
A
(5;
1.00 mg,
AHCTUNGTRENNUNG
0.86 mmol) was dissolved in THF (10 mL) and added dropwise to the
acid chloride mixture. After stirring for 8 h at ambient temperatures the
solvent was removed under reduced pressure and the crude product was
dissolved in CHCl3 (25 mL) and washed with a saturated solution of
NaHCO3 (25 mL) and then brine (25 mL). The organic solution was
dried over MgSO4 and the pure product obtained after precipitation with
CHCl3 and MeOH as a yellow powder in a yield of 86% (1.33 g,
0.74 mmol).
9
(831.0 mg, 0.25 mmol) was dissolved in formic acid (63 mL) and stirred
for 12 h. Then the solvent was removed and the product dried under re-
duced pressure to furnish the product as a beige powder in a yield of
91% (605.0 mg, 0.23 mmol).
1H NMR (CDCl3, 400 MHz, RT): d=0.87 (m, 12H; CH3), 1.28–1.42 (m,
80H; CH2), 2.02 (m, 24H; CH2), 2.20 (m, 24H; CH2), 3.23 (d, 2J=
12.5 Hz, 4H; CH2), 3.94 (t, 3J=7.3 Hz, 8H; CH2), 4.48 (d, 2J=12.0 Hz,
4H; CH2), 7.34 (s, 8H; CHarom), 7.57 (s, 4H; NH), 7.82 (d, 3J=8.0 Hz,
8H; CHarom, benzyl), 7.92 (d, 3J=8.0 Hz, 8H; CHarom; benzyl); 13C NMR
(CDCl3, 100.5 MHz, RT): d=13.8 (CH3), 22.2, 26.1, 28.2, 29.0, 29.4, 29.5,
29.6, 29.7, 29.8, 30.0, 31.5 (CH2), 57.5 (Cq-NH), 75.2 (CH2-O), 121.3
(CHarom), 127.4 (CHarom, benzyl), 133.3 (Carom-NHCO), 134.2 (Carom-CO),
137.6 (Carom-CH2), 137.9 (Carom-CONH), 152.5 (Carom-O), 164.5, 166.1,
174.7 ppm (CO); MS (MALDI-TOF, cinnamic acid): m/z: 2689.4 [M+
3
1H NMR (CDCl3, 400 MHz, RT): d=0.86 (t, J=6.0 Hz, 12H; CH3), 1.30
(m, 72H; CH2), 1.89 (m, 8H; CH2), 3.16 (d, 2J=13.4 Hz, 4H; CH2), 3.88
(m, 20H; OCH2, OCH3), 4.45 (d, 2J=13.4 Hz, 4H; CH2), 7.00 (s, 8H;
CHarom), 7.72 (d, 3J=8.5 Hz, 8H; CHarom), 7.87 (d, 3J=8.3 Hz, 8H;
CHarom), 8.03 ppm (s, 4H; NH); 13C NMR (CDCl3, 100.5 MHz, RT): d=
14.0 (CH3), 22.6, 29.4, 29.7, 29.8, 29.82, 29.9, 30.0 (CH2), 30.2, (CH2-Ar),
31.1 (CH2), 52.3 (O-CH3), 75.4 (CH2-O), 121.4 (CHarom), 127.1 (CHarom
,
NaÀH]+;
elemental
analysis
calcd
65.89,
(%)
7.48,
for
4.15;
benzyl), 129.7 (Carom-NHCO), 131.6 (CHarom, benzyl), 132.6 (Carom-CO),
135.4 (Carom-CH2), 138.3 (Carom-CONH), 154.1 (Carom-O), 164.8, 166.2 ppm
(CO); MS (FAB, NBA): m/z: 1806 [M]+; elemental analysis calcd (%)
for C112.25H148D0.25N4O16·0.25CDCl3 (1836.49): C 73.41, H 8.15, N 3.05, O
13.94; found: C 73.19, H 8.19, N 3.18.
C148.3H200D0.3Cl0.9N8O36·0.3CDCl3 (2703.32):
C
H
N
found: C 65.70, H 7.52, N 4.37.
Synthesis of 1-(4-bromobutyl)pyrene (12): PPh3 (816.00 mg, 3.11 mmol)
was added to solution of 4-pyren-1-ylbutanol (11; 712.00 mg,
a
Synthesis
of
5,11,17,23-tetrakis(carboxyphenylcarbonylamino)-
2.60 mmol) and CBr4 (2.65 mg, 5.30 mmol) in CH2Cl2 (20 mL). After
6 min the reaction was quenched with Na2CO3 (55 mL). After extracting
the aqueous phase three times with CH2Cl2 (20 mL), the combined or-
ganic phases were washed with Na2CO3 (20 mL) twice. Column chroma-
tography with cyclohexane/EtOAc (96:4) furnished the light-sensitive
product in a yield of 70% (625.12, 1.80 mmol).
1H NMR (300 MHz, CDCl3, RT): d=2.01 (m, 4H; CH2), 3.35 (m, 2H;
CH2), 3.45 (m, 4H; CH2), 7.84 (d, 3J=7.9 Hz, 1H; CH), 7.99 (m, 3H;
CHarom), 8.14 (m, 4H; CHarom), 8.25 ppm (d, 3J=9.2 Hz, 1H; CH);
13C NMR (300 MHz, CDCl3, RT): d=30.2, 32.55, 32.6, 33.6 (CH2), 123.2,
124.7, 124.8, 124.9, 125.0, 125.04, 125.8, 126.6, 127.2 (CHarom), 127.3,
127.5, 128.6, 129.9, 130.8, 131.4, 136.0 ppm (Cq-Ar); MS (FAB, NBA):
m/z: 338 [M+H]+; elemental analysis calcd (%) for C20H17Br (337.25): C
71.23, H 5.08; found: C 71.32, H 5.12.
25,26,27,28-tetrakis(dodecyloxy)calix[4]arene (8): Compound 7 (1.30 g,
0.74 mmol) was dissolved in THF (50 mL). H2O (3 mL) and LiOH
(142.0 mg, 5, 92 mmol) were added to this mixture. After 6 h at 608C the
reaction mixture was neutralized with 1m HCl and then the solvent was
removed and the product filtered and washed with water. After drying
the product was obtained as a yellow powder in a yield of 95% (1.23 g,
0.70 mmol).
1H NMR (DMSO, 400 MHz, RT): d=0.90 (m, 12H; CH3), 1.31–1,47 (m,
72H; CH2), 2.04 (m, 8H; CH2), 2.94 (brs, 4H; OH), 3.22 (d, J=12.8 Hz,
4H; CH2), 3.98 (t, J=7.5 Hz, 8H; CH2), 4.55 (d, J=12.8 Hz, 4H; CH2),
7.33 (s, 8H; CHarom), 7.86–8.01 (m, CH, benzyl), 9.51 ppm (s, 4H; NH-
CO); 13C NMR (DMSO, 100.5 MHz, RT): d=14.6 (CH3), 23.7, 27.6, 30.6,
30.9, 31.0, 31.1, 31.2, 31.24, 31.3 (CH2), 31.4 (CH2-Ar), 33.1 (CH2), 76.5
(CH2-O), 121.4 (CHarom), 128.5 (CHarom, benzyl), 130.5 (Carom-NHCO),
2
3
2
Synthesis of 1-(4-trimethylammoniobutyl)pyrene bromide (10): 1-(4-Bro-
mobutyl)pyrene (12; 300.00 mg, 0.86 mmol) was dissolved in THF (5 mL)
and NMe3 (2 mL, 50%) was added, whereupon a yellow precipitate
formed. The mixture was stirred for 8 h and then the solvent was re-
moved. The crude product was reprecipitated with EtOH/Et2O. Filtration
and drying under reduced pressure furnished the product as a yellow
powder in a yield of 10%.
1H NMR (300 MHz, CDCl3, RT): d=1.69 (m, 2H; CH2), 1.83 (m, 2H;
CH2), 3.22 (s, 6H; CH3), 3.33 (t, 3J=7.3 Hz, 2H; CH2), 3.52 (m, 2H;
CH2) 7.80 (d, 3J=7.8 Hz, 1H; CH), 7.95 (m, 3H; CHarom), 8.09 (m, 4H;
CHarom), 8.17 ppm (d, 3J=9.2 Hz, 1H; CH); 13C NMR (300 MHz, CDCl3,
RT): d=22.6, 27.7, 32.4 (CH2), 53.2 (CH3), 66.4 (CH2-NH), 123.2, 124.9,
125.0, 125.05, 125.1, 126.1, 126.9, 127.4 (CH-Py), 127.5, 127.7, 128.6,
134.2 (CHarom, benzyl), 134.7 (Carom-CO), 135.8 (Carom-CH2), 140.5 (Carom
-
CONH), 154.1 (Carom-O), 165.1, 167.5 ppm (CO); elemental analysis
calcd (%) for C109H140DCl3N4O165·CDCl3 (1870.67): C 69.98, H 7.65, N
3.00; found: C 69.73, H 7.71, N 3.31.
Synthesis of Newkome-type [G-1] 5,11,17,23-tetrakis(tert-butoxycarbo-
nylphenylcarbonylamino)-25,26,27,28-tetrakis(dodecyloxy)calix[4]arene
(9): Compound 8 (875.1 mg, 0.5 mmol) was dissolved in DMF (35 mL)
and the solution cooled to 08C. Then N’-(3-dimethylaminopropyl)-N-
ethylcarbodiimide (EDC; 479.3 mg, 2.5 mmol), 4-dimethylaminopyridine
(DMAP; 365.8 mg, 2.5 mmol), and 1-hydroxybenzotriazole (HOBt;
332.8 mg, 2.5 mmol) were added and the reaction mixture was stirred for
30 min. The first-generation Newkome dendrimer (1.66 g, 4 mmol) was
Chem. Eur. J. 2009, 15, 1637 – 1648
ꢂ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
1647

A. Hirsch, C. Bçttcher et al.
130.1, 130.8, 131.4, 135.0 ppm (Cq-Ar); MS (FAB, NBA): m/z: 316
[MÀBr]+; elemental analysis calcd (%) for C23H26N (396.36): C 67.70, H
6.61, N 3.53; found: C 69.78, H 6.55, N 3.51.
[12] The straight lines were linear fitted to the linear slopes at low (8ꢄ
10À6 to 8ꢄ10À13 molLÀ1) and high (1ꢄ10À4 to 9ꢄ10À4 molLÀ1) con-
centrations of 4 using the program origin. This method was estab-
lished to quantify the fluorescence quenching induced by the inter-
action between 4 and the pyrene derivatives.
[13] a) A. Bernheim-Groswasser, R. Zana, Y. Talmon, J. Phys. Chem. B
2000, 104, 4005; b) P. R. Majhi, P. L. Dubin, X. Feng, X. Guo, J.
Phys. Chem. B 2004, 108, 5980.
Acknowledgements
[14] The final resolution achieved in the reconstruction of 11041 individ-
ual aggregates of 4 was determined by the Fourier shell correlation
method (FSC) (see ref. [14a]) and amounts to about 9.8 ꢆ on the
basis of the 3s threshold criterion (ꢀ13.7 ꢆ at 0.5 criterion) and the
D2 point-group symmetry (see ref. [14b]); a) G. Harauz, M. van
Heel, Optic 1986, 73, 146; b) E. V. Orlova, P. Dube, J. R. Harris, E.
Beckmann, F. Zemlin, J. Markl, M. v. Heel, J. Mol. Biol. 1997, 271,
417.
[15] J.-K Kim, E. Lee, Y. B. Lim, M. Lee, Angew. Chem. 2008, 120, 4818;
Angew. Chem. Int. Ed. 2008, 47, 4740.
[16] a) K. Kalyanasundaram, J. K. Thomas, J. Am. Chem. Soc. 1977, 99,
2039; b) K. Kalyanasundaram, J. K. Thomas, J. Phys. Chem. Soc.
1977, 81, 2176.
[17] G. M. L. Consoli, G. Granata, R. Lo Nigro, G. Malandrino, C.
Geraci, Langmuir 2008, 24, 6194.
[18] G. Pistolis, A. Malliaris, D. Tsiourvas, C. M. Paleos, Chem. Eur. J.
1999, 5, 1440.
[19] The cmcs of 10 and 13 were determined by diluting them in buffer
at the corresponding pH. Plotting the development of the intensity
I1 of the pyrene moiety versus the concentrations of 10 and 13 re-
vealed a nonlinear concentration dependence with the concentration
at the maximum intensity representing the cmc.
We thank the Deutsche Forschungsgemeinschaft (B0 1000/6-1 to C.B and
HI 468/13-2 as well as Cluster of Excellence: Engineering of Advanced
Materials to A.H.) for financial support.
[1] a) M. Kellermann, W. Bauer, A. Hirsch, B. Schade, K. Ludwig, C.
Boettcher, Angew. Chem. 2004, 116, 3019; Angew. Chem. Int. Ed.
2004, 43, 2959; b) M. Kellermann, W. Bauer, A. Hirsch, B. Schade,
K. Ludwig, C. Bçttcher, Angew. Chem. 2004,116, 3019; Angew.
Chem. Int. Ed. 2004, 43, 2959.
[2] Y. Chang, M. Kellermann, M. Becherer, A. Hirsch, C. Bohne, Pho-
tochem. Photobiol. Sci. 2007, 6, 525.
[3] a) S. Burghardt, A. Hirsch, B. Schade, K. Ludwig, C. Bçttcher.
Angew. Chem. 2005, 117, 3036; Angew. Chem. Int. Ed. 2005, 44,
2976; Angew. Chem. Int. Ed. 2005, 44, 2976.
[4] U. Hartnagel, D. Balbinot, N. Jux, A. Hirsch. Org. Biomol. Chem.
2006, 36, 1785; a) B. Schade, K. Ludwig, C. Bçttcher, U. Hartnagel,
A. Hirsch. Angew. Chem. 2007, 119, 4482; b) B. Schade, K. Ludwig,
C. Bçttcher, U. Hartnagel, A. Hirsch, Angew. Chem. 2007, 119,
4472; Angew. Chem. Int. Ed. 2007, 46, 4393.
[5] S. Lucht, J. Stumpe, M. Rutloh, J. Fluorescence 1998, 8, 153.
[6] J. Heldt, D. Gormin, M. Kasha, Chem. Phys. Lett. 1988, 150, 433.
[7] F. Sansone, M. Dudic, G. Donofrio, C. Rivetti, L. Baldini, A. Cas-
nati, S. Cellai, R. Ungaro, J. Am. Chem. Soc. 2006, 128, 14528.
[8] K. Furuta, K. Tomokiyo, M. T. Kuo, T. Ishikawa, M. Suzuki, Tetrahe-
dron 1999, 55, 7529.
[20] M. Brettreich, A. Hirsch, Synlett 1998, 1396.
[21] D. Gutsche, M. Iqbal, Org. Synth. 1989, 68, 234.
[22] F. W. Kꢁster, A. Thiel, Rechentafeln fꢀr die Chemische Analytik,
102nd ed., de Gryter, Berlin, New York, 2002.
[9] G. P. L’Heureux, M. Fragata, Biophys. Chem. 1988, 30, 293.
[10] The benzamide intensity (IBA) was normalized to the highest mea-
sured intensity (IBA,max) at the corresponding pH value.
[11] E. Fuguet, C. Rafols, M. Roses, E. Bosch, Anal. Chim. Acta 2005,
548, 95.
Received: September 29, 2008
Published online: January 2, 2009
1648
www.chemeurj.org
ꢂ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2009, 15, 1637 – 1648