Fibrous networks with incorporated macrocycles: A chiral stimuli-responsive supramolecular supergelator and its application to biocatalysis in organic media

Article abstract of DOI:10.1002/chem.201300193

A new and versatile, crown ether appended, chiral supergelator has been designed and synthesized based on the bis-urea motif. The introduction of a stereogenic center improved its gelation ability significantly relative to its achiral analogue. This low-molecular-weight gelator forms supramolecular gels in a variety of organic solvents. It is sensitive to multiple chemical stimuli and the sol-gel phase transitions can be reversibly triggered by host-guest interactions. The gel can be used to trap enzymes and release them on demand by chemical stimuli. It stabilizes the microparticles in Pickering emulsions so that enzyme-catalyzed organic reactions can take place in the polar phase inside the microparticles, the organic reactants diffusing through the biphasic interface from the surrounding organic phase. Because of the higher interface area between the organic and polar phases, enzyme activity is enhanced in comparison with simple biphasic systems. Supramolecular gels: A versatile, crown ether substituted, chiral bis-urea gelator forms gels in a variety of organic solvents. The gel is sensitive to multiple chemical stimuli and the reversible sol-gel phase transitions can be triggered by host-guest interactions. The supramolecular gel can be used for enzyme immobilization and stimuli-induced release (see figure). When used in Pickering emulsions containing nanoparticle-stabilized gel microparticles with a polar inner phase, the gelator broadens the range of solvents in which enzymes can be used to catalyze reactions in nonpolar media. Copyright

Full text of DOI:10.1002/chem.201300193

DOI: 10.1002/chem.201300193
Fibrous Networks with Incorporated Macrocycles: A Chiral
Stimuli-Responsive Supramolecular Supergelator and Its Application to
Biocatalysis in Organic Media
Zhenhui Qi,[a] Changzhu Wu,[a] Paula Malo de Molina,[b] Han Sun,[c] Andrea Schulz,[a]
Christian Griesinger,[c] Michael Gradzielski,[b] Rainer Haag,[a]
Marion B. Ansorge-Schumacher,[b, d] and Christoph A. Schalley*[a]
Dedicated to Professor Dr. Jçrn Manz on the occasion of his 65th birthday
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Chem. Eur. J. 2013, 19, 10150 – 10159

FULL PAPER
Abstract: A new and versatile, crown
ether appended, chiral supergelator has
been designed and synthesized based
on the bis-urea motif. The introduction
of a stereogenic center improved its ge-
lation ability significantly relative to its
achiral analogue. This low-molecular-
weight gelator forms supramolecular
gels in a variety of organic solvents. It
is sensitive to multiple chemical stimuli
and the sol–gel phase transitions can
be reversibly triggered by host–guest
interactions. The gel can be used to
trap enzymes and release them on
demand by chemical stimuli. It stabiliz-
es the microparticles in Pickering emul-
sions so that enzyme-catalyzed organic
reactions can take place in the polar
phase inside the microparticles, the or-
ganic reactants diffusing through the
biphasic interface from the surrounding
organic phase. Because of the higher
interface area between the organic and
polar phases, enzyme activity is en-
hanced in comparison with simple bi-
phasic systems.
Keywords: biocatalysis
·
crown
compounds · gels · host–guest sys-
tems · self-assembly
Introduction
with a molecular recognition unit attached to the fibers,
which, however, is not directly involved in gel formation.[6]
Chemically triggered gel–sol transitions form the basis of
functional materials[7] with potential for drug delivery and
cosmetics:[8] A load trapped inside the cavities of a gel can
be liberated on demand when a suitable chemical signal in-
duces the gel–sol transition. Making two stimuli interde-
pendent like the inputs of a logic gate can help define much
more precisely under which conditions the gel–sol transition
occurs.[9] Function can also be based on the mechanical
properties of the gel, for example, by using the gel as a
matrix for immobilizing biocatalysts.[10] As most enzymes
operate best in water or polar solvents, their use in organic
reactions is often limited unless the phase incompatibility is
appropriately dealt with. This can be achieved either in
emulsions that are stabilized by amphiphilic surfactants or
in Pickering emulsions[11,12] in which the enzyme-containing
polar-phase droplets are stabilized by surrounding SiO2
nanoparticles. Gelling of the polar phase inside these nano-
particle-surrounded droplets stabilizes them with the syn-
thetic advantage that the droplets containing the enzyme
can easily be reused by separating them from the reaction
mixture by centrifugation.
Natural fibrous networks such as microtubuli or actin fila-
ments are self-assembled noncovalent architectures that are
essential for cytoskeleton construction and give cells their
shape.[1] Highly dynamic in assembly and disassembly, the
fibers are equipped with sites for the recognition of, for ex-
ample, the kinesin linear motors that, driven by adenosine
triphosphate (ATP) hydrolysis, actively transport carriage
along the microtubuli across the cell.[2] Beyond their archi-
tectural role expressed in terms of cell shape, the fibrous
network is thus also an integral part of the metabolism and
behavior of the cell. Such function not only requires a high
degree of hierarchical order, but also selective responsive-
ness to chemical signals in the microenvironment that con-
trols the metabolismꢁs complex spatio-temporal pattern de-
velopment.
Synthetic fibrous networks in supramolecular gels[3] cer-
tainly do not yet match the complexity and functional preci-
sion of their natural counterparts but can nevertheless be
functional. On the one hand, their noncovalent nature
makes them responsive[4] to chemical stimuli that compete
with the binding interactions between the individual gelator
molecules[5] and thus control the assembly or disassembly of
gel fibers. More challenging, on the other hand, is to imple-
ment gelation control through interactions of the stimulus
Results and Discussion
Gelator synthesis and gel formation: The two enantiomers
of the chiral low-molecular-weight gelator, (R)-1 and (S)-1
(Scheme 1), are bis-urea gelators[13] equipped with a benzo-
21-crown-7 ether.[14] The crown ether represents a molecular
recognition unit that makes the gelator susceptible to a vari-
ety of different chemical stimuli, presumably without being
directly involved in gel formation. The cavity size of the
crown is not only suitable for K+ binding, but also allows di-
alkylammonium ions to form pseudorotaxanes by threading
through the crown ether.[15] As a third chemical stimulus,
competitive anions such as chloride can interfere with the
urea–urea hydrogen bonding between adjacent gelator mol-
ecules.[16]
[a] Z. Qi, Dr. C. Wu, A. Schulz, Prof. R. Haag, Prof. C. A. Schalley
Institut fꢂr Chemie und Biochemie, Freie Universitꢃt Berlin
Takustr. 3, 14195 Berlin (Germany)
Fax : (+49)30-838-55817
E-mail: christoph@schalley-lab.de
[b] Dr. P. Malo de Molina, Prof. M. Gradzielski,
Prof. M. B. Ansorge-Schumacher
Institut fꢂr Chemie, Technische Universitꢃt Berlin
Straße des 17. Juni 124, 10624 Berlin (Germany)
[c] H. Sun, Prof. C. Griesinger
Max-Planck-Institut fꢂr Biophysikalische Chemie
Am Fassberg 11, 37073 Gçttingen (Germany)
[d] Prof. M. B. Ansorge-Schumacher
Current address: Institut fꢂr Mikrobiologie
Technische Universitꢃt Dresden, 01062 Dresden (Germany)
The gelator enantiomers (R)-1 and (S)-1 were synthesized
as summarized in Scheme 1. Mono-Boc-protected 1,4-bis-
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201300193.
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C. A. Schalley et al.
Table 1. Results of the gelation experiments with compound (R)-1.[a]
Solvent
State
CGC[b]
n-butanol
n-propanol
ethanol
gel
gel
gel
gel
gel
gel
gel
gel
gel
0.75 wt.%, 6 mgmLꢀ1
0.88 wt.%, 7 mgmLꢀ1
1.64 wt.%, 13 mgmLꢀ1
1.25 wt.%, 10 mgmLꢀ1
0.63 wt.%, 5 mgmLꢀ1
0.38 wt.%, 3 mgmLꢀ1
0.63 wt.%, 5 mgmLꢀ1
0.38 wt.%, 3 mgmLꢀ1
0.10 wt.%, 0.8 mgmLꢀ1
methanol
dichloromethane
chloroform
1,4-dioxane
nitromethane
acetonitrile
DMSO, DMF
solution
partial gel[c]
partial gel[c]
partial gel[c]
insoluble
ethyl acetate, n-butyl acetate
tetrahydrofuran, isobutyl alcohol
toluene, xylene
cyclohexane, n-heptane, dodecane
diethyl ether, acetone, water
insoluble
[a] All spectra were recorded at a concentration of 14 mgmLꢀ1. [b] For
the gels, the 208C critical gelation concentrations (CGC) are given. The
CGC is defined as the concentration below which no gel forms. [c] The
term partial gel refers to a very viscous fluid.
Scheme 1. Synthesis of chiral gelators (R)-1 and (S)-1. Achiral bis-crown
gelator 7 was prepared and characterized similarly as reported earlier.[9b]
It serves as a comparison here.
gelator (R)-1/(S)-1 is more versatile in terms of solvent
choice and has a higher gelation ability than previously re-
ported achiral gelator 7 bearing two benzo-21-crown-7
groups.[9b]
ꢀ
FTIR experiments on the gels revealed N H stretch vi-
(ami
E
brations at around 3318 cmꢀ1 and amide bands at around
1616 and 1568 cmꢀ1, which are typical of hydrogen-bonded
urea groups (Table 2). Urea–urea hydrogen-bonding is thus
urea derivatives (R)-2 and (S)-2, which were subsequently
deprotected to yield (R)-3 and (S)-3. To complete the gela-
tor synthesis and incorporate the second urea moiety, the
crown ether was attached to the free amino group as the iso-
cyanate 6, which was synthesized from the corresponding
amine 4[9b] in situ. Although the synthesis of the gelator con-
tains two steps that give only mediocre yields, it is quite effi-
cient because the purification of all the intermediates and
the final product can be achieved simply by precipitation,
filtration, and washing. The elemental analysis of the final
gelator molecules confirmed that a high purity was achieved
without additional purification such as chromatography or
recrystallization. As the chiral center is not affected by any
of the synthetic procedures, the enantiomeric excess remains
unchanged with respect to that of the commercially availa-
ble (R)- or (S)-(ꢀ)-a-methylbenzyl isocyanate used as the
reactant (eeꢁ99%).
Table 2. FTIR data for (R)-1 in the gel and solid state.[a]
Solvent
Amide absorption bands [cmꢀ1
]
NH stretch
Amide-I
Amide-II
n-butanol
n-propanol
ethanol
n.d.[b]
n.d.[b]
n.d.[b]
n.d.[b]
3319.0
3313.4
3318.7
3317.7
3325.5
3318.7
1616.4
1616.7
1616.4
1616.0
1615.7
1615.4
1616.7
n.d.[c]
1568.1
1565.2
1564.4
1565.8
1568.3
1569.4
1566.6
n.d.[c]
methanol
dichloromethane
chloroform
1,4-dioxane
nitromethane
acetonitrile
solid state
1616.1
1615.4
1568.6
1565.3
[a] All spectra were recorded at a concentration of 14 mgmLꢀ1 at room
temperature. [b] Not detected because bands are superimposed by the
broad O H stretch vibration band of the solvent. [c] Not detected, be-
cause bands are superimposed by the symmetric and antisymmetric
stretch vibration bands of the nitro group of the solvent.
ꢀ
Enantiopure gelators (R)-1 or (S)-1 form gels (Table 1)
with quite low critical gel concentrations (CGC) in a variety
of different solvents, among them linear-chain alcohols,
chlorinated hydrocarbons (CH2Cl2 and CHCl3), 1,4-dioxane,
nitromethane, and, in particular, acetonitrile. In this last sol-
vent, (R)-1/(S)-1 can be regarded as supergelators as gela-
an important factor for gel formation.[17] As reported earli-
er,[18] an antiparallel arrangement of the two urea groups is
pivotal for gelation. We assume this to be also true for (R)-1
and (S)-1. On a macroscopic level, the gels of (R)-1 or (S)-1
in acetonitrile display typical thermoreversibility and are
characterized by somewhat concentration-dependent gel–sol
transition temperatures Tgs that range from 327 K for a gela-
tor concentration of 5.0 mm to 354 K for a concentration of
25.0 mm (see Figure S15 in the Supporting Information).
ACHTUNGTRENNUNGtion occurs at concentrations below 0.1 wt.%. In contrast,
the gelator is insoluble in nonpolar solvents such as linear or
cyclic alkanes and in aromatic hydrocarbons. At the other
end of the polarity scale are solvents with high hydrogen-
bond acceptor strengths such as dimethyl sulfoxide (DMSO)
or dimethylformamide (DMF), in which the gelator dis-
solves without gel formation. With these gelation properties,
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Fibrous Networks Incorporating Macrocycles
FULL PAPER
Gel morphology and its formation mechanism: Because
electron microscopy is problematic with acetonitrile as sol-
vent, atomic force microscopy (AFM) was used to character-
ize the gel morphology (Figure 1). The AFM images show
the formation of entangled networks of quite uniform, mi-
crometer-long, straight helical fibers in the gels of both
enantiomers, (R)-1 and (S)-1. The fibers strongly overlap in
the gel state so that dilute samples were spin-coated on to
mica to generate separate fibers suitable for a more detailed
analysis. Well-defined right-handed P helices originate from
(R)-1, whereas (S)-1 affords predominantly left-handed M
helices (as labeled in Figure 1). In some images, a few dia-
stereomeric fibers with the opposite helicity were also ob-
served. This indicates the kinetically controlled co-formation
of a minor amount of oppositely twisted fibers that are pre-
vented from converting into the predominant helicity once
they are formed by high activation barriers (see Figures S27
and S29 in the Supporting Information). Such a phenome-
ACHTUNGTRENNUNGnon was similarly observed in the self-assembly of gels
formed from chiral merocyanine dyes.[19] It also rationalizes
the nonperfect mirror symmetry of the CD and VCD spec-
tra (see Figure S16 in the Supporting Information) of the
two gelator enantiomers: Depending on the exact conditions
during gel formation, different amounts of the second type
of fiber may form. As the two fibers have a diastereomeric
relation to each other, the CD spectra may differ depending
on their ratio. Overall, the methyl group at the chiral center
exerts quite strong, but not perfect, control over the helicity
of the fibers. The predominant helicity observed is in agree-
ment with the results of semiempirical AM1 MOZYME cal-
culations performed on a hexadecameric aggregate (see Fig-
ure S13 in the Supporting Information).[20] In the calcula-
tions, the benzo-21-crown-7 ether was replaced by phenyl.
When the calculated helices turn as observed by experiment,
the methyl groups point sideways and thus find sufficient
space to avoid steric repulsion with the next neighboring ge-
lator molecule. If one performs a calculation on the opposite
helix, the methyl groups are directed towards the neighbor-
ing gelator molecule causing higher steric strain.
AFM section analyses revealed a fiber height of around
6–7 nm with a helical pitch of around 47 nm (Figure 1c,f and
Figures S32 and S33 in the Supporting Information). More
precise information regarding the fiber diameter was ob-
tained from small-angle neutron scanning (SANS) measure-
ments (Figure 1g), which showed the fibers to be very well
described by homogeneous cylinders with a diameter of
7.6ꢂ0.2 nm (simulated data for differing cross-section diam-
eters are given in Figure 1g and shows the substantial dis-
crepancies occurring for other assumed diameters). Only for
q<0.08 nmꢀ1 is a significant deviation of the experimental
scattering data from the theoretical curve observed, which
means that for sizes above 80–100 nm, deviations from a
simple cylindrical structure are present. This upturn at low q
can be ascribed to the formation of a network structure, in
agreement with the structure observed by AFM (Fig-
ure 1a,d). It can also be noted that overall the structure is
only little affected by the concentration in the studied range
Figure 1. a,d) AFM height images (1.6ꢄ1.6 mm) of samples prepared by
spin-coating solutions of a) (R)-1 and d) (S)-1 obtained by the dilution of
0.127 wt.% chiral gels in acetonitrile to 0.05 and 0.025 wt.%, respectively.
b,e) Enlarged AFM images of fibers formed from b) (R)-1 and e) (S)-1.
c,f) Height profiles along the red lines indicated in images (a) and (d).
g) SANS curves measured for gelator (R)-1 at concentrations of
1.80 wt.% (*) and 0.44 wt.% (&) as a function of the scattering vector
q in deuterated acetonitrile. The colored solid lines correspond to fits to
infinitely long cylinders with different cross-sections. Cylinders with di-
ameters of 7.4 nm give the best fit to the experimental data. The upturn
of the intensity at low q indicates network formation.
of 0.44–1.80 wt%. The somewhat smaller height determined
by AFM may be rationalized by flattening of the fibers
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C. A. Schalley et al.
during sample drying. The diameter of the fiber is conse-
quently about twice that of the calculated monomer length
of around 3.15 nm. Interestingly, in some AFM images, a
few thinner fibrils with a diameter of around 3.9 nm can be
observed (see Figure S25 in the Supporting Information).
This value is not too different from the length of a gelator
monomer. As proposed for gelator 7 previously,[9b] these
values indicate that the predominant fibers are bundles of
either two or four fibrils that form coils. Based on these re-
sults we suggest a hierarchical self-assembly process for gel
formation (Scheme 2): Initial nucleation of (R)-1 or (S)-1
Scheme 2. a) Structure of the gelator monomer optimized at the AM1
level of theory as implemented in the CaChe 5.0 program package.[20]
b) Schematic illustration of the hierarchical self-assembly of the gel. Nu-
cleation of the monomer into small oligomers is followed by the forma-
tion of fibrils that coil around each other to form fibers. The final step is
the formation of entangled networks that entrap the solvent to form the
gel.
into hydrogen-bonded dimers and small oligomers is fol-
lowed by a quick growth into long helical fibrils.[21] The fi-
brils can further coil around each other giving rise to helical
fibers that are about twice as thick. However, it remains un-
clear from the AFM and SANS data how many fibrils coil
around each other to form a fiber. Fibrils and bundles final-
ly entangle to form a gel and immobilize solvent molecules
inside its cavities.
Figure 2. a) Three different chemical stimuli that induce gel–sol transi-
tions: K+ binding, pseudorotaxane formation, and anion binding. The ef-
fects of all three stimuli can be reversed by scavenging the K+ with a
cryptand, deprotonation and subsequent deslipping of the ammonium ion
axle, and precipitation of the chloride as AgCl, respectively. b) Supramo-
lecular gel (R)-1 (3.6 wt.% in acetonitrile) and its stimuli-responsive be-
havior. Gel–sol transitions controlled by K+/
ACTHNUTRGNE[UNG 2.2.2]cryptand addition
(top), acid/base-controlled pseudorotaxane formation with G1 (middle),
and addition of AgCl (bottom). The letters identify the gels as used in
the rheology section. Gels are indicated by upside-down test-tubes that
show the formation of a solid sample. Sols are represented by horizontal
test-tubes that show the fluid behavior of the sample. CD spectral
changes during the progressive addition of c) K+ and d) G1. AFM ampli-
tude error images of the gel after addition of e) 0.25 equivalents of KPF6
and f) 0.5 equivalents of G1 showing the changes in morphology occur-
ring upon guest addition.
Responsiveness to chemical stimuli: The gel is sensitive to
the addition of three different chemical stimuli (Figure 2a).
The addition of 1 equiv KPF6 induces a gel–sol transition,
which can be reversed to the gel state by the addition of
[2.2.2]cryptand as K+ scavenger (Figure 2b, top row). This
1
gel–sol phase transition cycle can be followed by H NMR
spectroscopy (see Figure S28 in the Supporting Informa-
tion). As the monomer is CD-silent at wavelengths of 220
and 272 nm, CD spectroscopy can also be used to monitor
the gel–sol transition (Figure 2c). The CD signals of the heli-
ces are increasingly reduced with increasing K+ concentra-
tion. In a control experiment, NEt4PF6 did not induce a gel–
ure S36 in the Supporting Information). The gel remains
stable upon addition of up to 0.25 equiv KPF6, but its mor-
phology changes to fibers with a smaller helical pitch (Fig-
ure 2e). Upon addition of 1 equiv K+, nearly all the crown
ethers are occupied and charge repulsion likely drives the
monomers apart so that the fibrous structure is destroyed.
The second chemical signal that induces a gel–sol transi-
ꢀ
sol transition, which shows the weakly coordinating PF6
anion does not interfere with the gel structure (see Fig-
tion is the addition of benzylACTHNGUTRENNU(G n-butyl)ammonium hexafluo-
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Fibrous Networks Incorporating Macrocycles
FULL PAPER
ACHTUNGTRENNUNGroACHTUNGTRENNUNGphosphate (G1; Figure 2a). Again, the intensity of the
signal reveals the persistence or destruction of the gel (Fig-
ure 2b, middle row): The addition of 2 equiv does not de-
stroy the gel, but 3 equiv of G1 does. Secondary dialkylam-
monium ions with at least one unbranched alkyl chain are
known to slip through the cavity of benzo-21-crown-7 to
form pseudorotaxanes. However, dibenzylammonium does
not because the phenyl groups are too bulky to penetrate
the crown ether.[15] Thus, in contrast to G1, dibenzylammo-
nium hexafluorophosphate is unable to induce the gel–sol
transition in a control experiment (see Figure S39 in the
Supporting Information,). Thus, pseudorotaxane formation
is the reason behind the gel–sol transition mediated by G1
as the chemical stimulus. This is also supported by the
1H NMR spectra (see Figures S37 and S38), which exhibit
shifts in signals typical of the threading of G1 into benzo-21-
crown-7.[9b,15,22] However, only some of the crown ethers
appear to be occupied by axles, as signals from free ammo-
nium ions are still observed. This may explain why more
than 1 equivalent of the ammonium hexafluorophosphate is
required to break the gel. Reversible gel–sol and sol–gel
transitions are achieved by the addition of triethylamine
(NEt3); the ammonium ion is deprotonated leading to the
dissociation of the pseudorotaxanes and thus restoration of
the gel. Subsequent addition of trifluoroacetic acid (TFA)
regenerates the ammonium ion, which then slips into the
cavity of the crown ether thereby causing destruction of the
gel again.
Figure 3. a,b) Storage modulus G’ and loss modulus G’’ for various gel
samples (gelator concentration 41.6 mm in acetonitrile) as a function of
oscillatory frequency at 258C. c) G’/G’’ ratio for the different gel samples
as a function of frequency. The letters identify the gels as defined in
Figure 2.
Finally, chloride ions interfere with the urea–urea hydro-
gen bonding interactions (Figure 2b, bottom row).[16] Again,
a substantial amount of chloride ions (8 equiv of NEt4Cl)
are required to induce the gel–sol transition, which indicates
a significant stability of the gel. This gel–sol transition can
be reversed by the addition of a stoichiometric amount of
AgPF6, which causes the precipitation of the chloride, but
cally 9.5 nm. This is much smaller (by about a factor of 10)
than the overlap distance deduced from SANS, which means
that for such an overlapping network of fibers, a much
higher energy than kT is stored per structural unit, which
can be explained by the high energy required to deform and
bend the rather thick fibers.
ꢀ
the weakly coordinating PF6 counter ion does not interfere
with the gel formation. The resulting NEt4PF6 side-product
has no significant effect on gel formation, but the gel is
more opaque due to presence of the AgCl precipitate. Thus,
three different chemical stimuli can be used to induce gel–
sol transitions and each of them can be reversed by the ap-
propriate reagents. This opens the way to the construction
of logic gates in a similar way to that described earlier for
the bis-crown analogue 7.[9b]
G0 ¼ 1NkT
ð1Þ
The loss modulus G’’ that accounts for the viscous proper-
ties of the gel is always substantially smaller, but in general
changes in parallel with G’ as a function of frequency.
Therefore the relative elasticity, as quantified by the ratio
G’/G’’, is fairly constant as a function of frequency. The orig-
inal (R)-1 gel has the highest value (ca. 7 at 1 Hz), and is
thus the most elastic gel, the values for the other gels gener-
ally ranging from 2 to 7 (Figure 3c). This means that all the
gels investigated also have a substantial viscous component,
as is typically observed in such self-assembled gels.
Rheological characterization of original and switched gels:
All the gels investigated generally show rheological features
similar to the original (R)-1 gel (Figure 3). Typically, the
storage modulus G’ is in the range of 2–5 kPa, increasing
with frequency as is typically observed for organogels.[23]
Only for gel G is a higher value of around 10 kPa observed,
whereas gels B and F are significantly weaker, with values
of about 200–500 Pa. The high frequency limit of the storage
modulus, the shear modulus G0, can then be related to the
number density 1N of elastic connecting points of the gel
through Equation (1). From this expression, we can deduce
for a G0 of 5 kPa that the network units have a size of typi-
Enzyme immobilization in Pickering emulsions: To show
the utility of the gelator under study, we provide here a
proof-of-principle for the immobilization of an enzyme[24] in
Pickering emulsions (Figure 4a). The aim was to devise a re-
liable way to deal with phase incompatibility when enzymes
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C. A. Schalley et al.
time by measuring the change in fluorescence intensity at
l=518 nm (excitation wavelength: 495 nm). Comparison
with a control, to which no KPF6 was added, clearly showed
a rapid release of the enzyme in the first few minutes,
whereas almost no change was observed in the control.
Thus, enzyme release can be efficiently induced by the addi-
tion of potassium ions.
Following these experiments that showed the gel of (R)-1
to be capable of immobilizing FITC-CalB and releasing it
by means of chemical stimulus, the gelator was used for the
preparation of Pickering emulsions. Silica-nanoparticle-sta-
bilized, FITC-CalB-loaded acetonitrile/water (9:1) droplets
were jellified in hexane with (R)-1 as described below. Con-
focal laser scanning microscopy (CLSM) fluorescence imag-
ing (Figure 5a) demonstrated the successful formation of the
emulsion. Green fluorescent microparticles were formed
with diameters in the range of 5–10 mm, with no fluores-
cence being observed from the surrounding hexane phase.
The enzyme is thus exclusively located in the polar-phase
microparticles. Figure 5a clearly shows that the enzyme is
distributed nonhomogeneously within the microparticles,
which can be attributed to physical adsorption on the gel
network.[25] Centrifugation (6000 rpm, 10 min) produced a
fluorescent sediment with a nonfluorescent hexane phase,
which proves that the droplets can be removed from the or-
ganic phase very easily (Figure 5b). As the droplets are
stable and do not lose their integrity during centrifugation,
they can be reused a multiple number of times. Figure 5c,d
shows the situation after the addition of K+ to the Pickering
emulsion. Clearly the droplets release FITC-CalB when the
gel inside turns into a sol and the trapped enzyme can be re-
gained after gentle centrifugation (6000 rpm, 30 s). K+-in-
duced enzyme release has again been followed over time
and compared with a control experiment in which no potas-
sium ions were added. As noted above for the gel, the in-
crease in fluorescence intensity in the supernatant solution,
and with it the enzyme release, is much faster when the gel
is destroyed by the addition of K+ (Figure 5e).
The CD spectra of the native enzyme and that released
and recollected from the microparticles (Figure 5f) are very
similar and exhibit the typical double absorbance at 208 and
220 nm indicative of a-helices that are a prominent structur-
al feature in CalB. The similarity of both CD spectra indi-
cates no or only very minor structural differences between
the native enzyme and that released from the emulsion mi-
croparticles. Consequently, K+ can also be used as a trigger
to release the immobilized enzymes from the droplets in
Pickering emulsions in which the enzyme is likely trapped in
its native conformation. To the best of our knowledge, this
represents the first example of the use of supramolecular
gels in Pickering emulsions for controlled enzyme immobili-
zation and release.
Figure 4. a) Schematic illustration of a Pickering emulsion as used for en-
zymatic catalysis of organic reactions in organic media. The inner polar
phase II contains the enzyme trapped in jellified microparticles that are
stabilized by SiO2 nanoparticles. b) Gel of (R)-1 loaded with FITC-CalB.
c) Enzyme release upon addition of KPF6 to the gel. d) Enzyme release
with time from a gel after addition of KPF6 (red circles) and in a control
without added KPF6 (black squares).
are used for organic reactions that proceed in quite nonpo-
lar organic media. The principle is shown in Figure 4a. The
enzyme is trapped within jellified microparticles formed of a
polar phase (here acetonitrile/water, 9:1, v/v), which are sta-
bilized by surrounding silica nanoparticles. The microparti-
cles are then immersed in an nonpolar organic phase (here
hexane), which contains the reactants for the enzyme-cata-
lyzed reactions. As a result of the large surface area be-
tween the two phases, efficient enzyme catalysis is expected
to occur within the polar phase inside the particles. Further-
more, the stimuli-responsiveness of the gelator should
render the gel itself as well as the carrier systems of the
Pickering emulsions capable of controlled enzyme release.
For the proof-of-principle experiment described here, lipase
B from Candida antarctica labeled with fluorescein isothio-
cyanate (FITC-CalB) was used. The test reaction is the con-
densation of 1-octanol and octanoic acid to yield the corre-
sponding ester.
Before investigating the Pickering emulsions, enzyme im-
mobilization and release was tested simply in a gel of (R)-1
prepared in the presence of FITC-CalB in acetonitrile/water
(9:1) by sonication (Figure 4b). The addition of water to the
gel followed by vortexing the sample for 2 min did not
result in a significant release of the entrapped FITC-CalB
because the layer of water remained fluorescent-silent. The
gel decomposed and released the enzyme upon addition of
KPF6, as indicated by the clearly visible green fluorescence
distributed in the water phase above the gel (Figure 4c). To
follow the enzyme release quantitatively (Figure 4d), the
change in concentration of FITC-CalB in the water phase
on top of the gel after addition of KPF6 was followed over
The activity of the immobilized enzyme was studied in the
esterification of 1-octanol and 1-octanoic acid as a model re-
action (Figure 5g,h). The specific activity of the lipase immo-
bilized in the gelled Pickering emulsion microparticles
(65.70 Umgꢀ1) is almost 20 times greater than that of the
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Fibrous Networks Incorporating Macrocycles
FULL PAPER
same amount of native enzyme in a liquid water/hexane bi-
phasic system (3.37 Umgꢀ1) containing the same overall
amount of water as that used to prepare the Pickering emul-
sions. To include a control that contains exactly the same
amounts of all the solvents used in the experiments with the
Pickering emulsions, this experiment was repeated in a bi-
phasic system containing hexane, water, and acetonitrile. In
this case the specific activity is somewhat higher than in the
water/hexane system, but still lower by a factor of four than
that obtained in the Pickering emulsions. This increased spe-
cific activity can be attributed to the considerably larger in-
terfacial area of the SiO2-nanoparticle-stabilized microparti-
cles, which improves the mass transfer between the two
phases and the accessibility to the enzyme catalyst.[12,26] In-
terestingly, different amounts of lipase in the gel particles
yield almost the same specific activity (Figure 5g). Also, the
initial reaction rates of the esterification reaction increase
with enzyme concentration. This reflects the open network
structure of the gel and the low resistance to the diffusion of
the rather small substrate molecules within the particles
compared with the much larger enzyme molecules, which
are efficiently trapped. These experiments show the utility
of gelator (R)-1 in enzyme-catalyzed reactions in organic
media.
Conclusion
A low-molecular-weight bis-urea gelator has been prepared,
the chirality of which determines the helicity of the fibers
formed during gelation. It is versatile and forms gels in a
number of different solvents with quite low critical gelation
concentrations. In acetonitrile, it can even be regarded as a
supergelator as the CGC is less than 0.1 wt.%. The gelator
is substituted with a crown ether making it susceptible to
different chemical stimuli that induce gel–sol transitions: K+
ion binding, pseudorotaxane formation between dialkylam-
monium ions and the crown ether, and interference of the
urea–urea hydrogen bonds through the interactions with
chloride anions. Each of these stimuli can be reversed such
that the gel–sol transitions are reversible. Enzymes such as
lipase B from Candida antarctica can be immobilized within
the gel and released on demand by the addition of a chemi-
cal stimulus, as exemplarily shown with K+. The gelator is
the first low-molecular-weight gelator used for enzyme im-
mobilization in Pickering emulsions. It thus contributes to
solving the phase problem that often exists when enzymes
are to be used to catalyze organic reactions in nonpolar or-
ganic media. The catalytic activity of CalB in Pickering
emulsions is significantly higher than that in the correspond-
ing biphasic solvent system. As the microparticles are stable
enough to withstand centrifugation, the catalyst can easily
be separated from the reaction mixture.
Figure 5. a) Fluorescence microscopy image of jellified acetonitrile/water
(9:1) microparticles loaded with FITC-CalB and stabilized by 140 nm
SiO2 nanoparticles (nonpolar phase I: hexane; polar phase II: water/ace-
tonitrile, 9:1; conc. of (R)-1 in phase II: 1.8 wt.%; conc. of SiO2-NP:
1.0 wt.%). Inset: Enlarged CLSM image of a jellified FITC-CalB micro-
particle. b) Jellified Pickering emulsion droplets loaded with FITC-CalB
as a fluorescent sediment in hexane after centrifugation. c) Release of
FITC-CalB from the microparticles induced by decantation of the
hexane phase and KPF6 addition. d) Released enzyme in the supernatant
after centrifugation. e) Changes in fluorescence intensity in the superna-
tant above the microparticles after centrifugation at 6000 rpm for 10 min
and addition of 15 mg KPF6 in water (red circles) or water alone (black
squares). f) CD spectra of native CalB (red line) and CalB released from
jellified Pickering emulsion droplets (black line). g) Specific activities of
CalB (grey) in biphasic hexane/water systems with or without acetonitrile
as control (amounts identical to the amounts used in the experiment with
Pickering emulsions) and CalB immobilized in Pickering emulsions (red
line). h) Linear dependence of the initial conversion of substrates with
time at different concentrations of enzymes in the emulsion microparti-
cles.
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C. A. Schalley et al.
Acknowledgements
Experimental Section
We thank the Deutsche Forschungsgemeinschaft for financial support
(SCHA 893/5 and SFB 765). Z.Q. is grateful to the China Scholarship
Council (CSC) for a Ph. D. fellowship. We thank Leonardo Chiappisi for
performing the SANS measurements. We acknowledge Marie-Sousai Ap-
pavou (JCNS) for help with the SANS experiments and the JCNS for
granting beamtime.
Gelation experiments and stimuli responsiveness of the gels: The gelator
and the solvent were placed in a capped test-tube, sonicated for 3 min,
and if necessary heated until the solid dissolved. The sample vial was
cooled to 258C and left to stand for 1 h under ambient conditions. The
state was evaluated by the “stable to inversion of a test-tube” method.
Gel–sol transition temperatures Tgs were determined by the “dropping-
ball method”.[27] A small ball (166 mg) was placed on top of the gel in a
test-tube (internal diameter 1.0 cm), which was slowly heated in a water
bath at a rate of 1 Kminꢀ1. Tgs is defined as the temperature at which the
ball reaches the bottom of the test-tube. Dropping-ball experiments were
carried out at least in duplicate, and the Tgs values obtained were repro-
ducible to within ꢂ18C. To examine the guest-induced stimuli responsive
behavior, the guest compound was added to the test-tube containing the
already-prepared gel. Subsequently, the capped test-tube was sonicated
for 5 min. The sample vial was left to stand for 15 min under ambient
conditions. The state was evaluated as described above.
[1] D. A. Fletcher, R. D. Mullins, Nature 2010, 463, 485–492.
[2] S.-i. Tamaru, M. Ikeda, Y. Shimidzu, S. Matsumoto, S. Takeuchi, I.
Hamachi, Nat. Commun. 2010, 1, 20.
[3] For selected recent reviews, see: a) N. M. Sangeetha, U. Maitra,
Chem. Soc. Rev. 2005, 34, 821–836; b) R. G. Weiss, Acc. Chem. Res.
2006, 39, 489–497; c) P. Dastidar, Chem. Soc. Rev. 2008, 37, 2699–
2715; d) L. E. Buerkle, S. J. Rowan, Chem. Soc. Rev. 2012, 41, 6089–
6102; e) A. Noro, M. Hayashi, Y. Matsushita, Soft Matter 2012, 8,
2416–2429; f) D. K. Smith in Supramolecular Chemistry: From Mol-
ecules to Nanomaterials (Eds.: P. A. Gale, J. W. Steed), Wiley, Hobo-
ken, 2012, pp. 3355–3376; g) H. Svobodovꢅ, V. Noponen, E. Koleh-
mainen, E. Sievanen, RSC Adv. 2012, 2, 4985–5007; h) S. Seiffert, J.
Sprakel, Chem. Soc. Rev. 2012, 41, 909–930; i) D. Das, T. Kar, P. K.
Das, Soft Matter 2012, 8, 2348–2365; j) E. A. Appel, J. del Barrio,
X. J. Loh, O. A. Scherman, Chem. Soc. Rev. 2012, 41, 6195–6214.
[4] For examples of gels responsive to different stimuli, see: a) T.
Ogoshi, Y. Takashima, H. Yamaguchi, A. Harada, J. Am. Chem.
Soc. 2007, 129, 4878–4879; b) H. Maeda, Chem. Eur. J. 2008, 14,
11274–11282; c) G. O. Lloyd, J. W. Steed, Nat. Chem. 2009, 1, 437–
442; d) J. H. Kim, M. Seo, Y. J. Kim, S. Y. Kim, Langmuir 2009, 25,
1761–1766; e) T. Tazawa, S. Yagai, Y. Kikkawa, T. Karatsu, A. Kita-
mura, A. Ajayaghosh, Chem. Commun. 2010, 46, 1076–1078; f) J. A.
Foster, J. W. Steed, Angew. Chem. 2010, 122, 6868–6874; Angew.
Chem. Int. Ed. 2010, 49, 6718–6724; g) Y. Suzaki, T. Taira, K. Osa-
kada, J. Mater. Chem. 2011, 21, 930–938; h) D. Dꢆaz Dꢆaz, D. Kꢂh-
beck, R. J. Koopmans, Chem. Soc. Rev. 2011, 40, 427–448; i) Y.-S.
Su, J.-W. Liu, Y. Jiang, C.-F. Chen, Chem. Eur. J. 2011, 17, 2435–
2441; j) X. Wang, L. Zhou, H. Wang, Q. Luo, J. Xu, J. Liu, J. Colloid
Interface Sci. 2011, 353, 412–419; k) F. Rodrꢆguez-Llansola, J. F. Mir-
avet, B. Escuder, Chem. Commun. 2011, 47, 4706–4708; l) V.
Lozano, R. Hernandez, A. Arda, J. Jimenez-Barbero, C. Mijangos,
M.-J. Perez-Perez, J. Mater. Chem. 2011, 21, 8862–8870; m) L.
Chen, Y.-K. Tian, Y. Ding, Y.-J. Tian, F. Wang, Macromolecules
2012, 45, 8412–8419; n) L. Tan, Y. Liu, W. Ha, L.-S. Ding, S.-L.
Peng, S. Zhang, B.-J. Li, Soft Matter 2012, 8, 5746–5749.
[5] For selected examples, see: a) X. Liao, X. Chen, G. Chen, X, Liu,
W. Chen, F. Chen, M. Jiang, Angew. Chem. 2010, 122, 4511–4515;
Angew. Chem. Int. Ed. 2010, 49, 4409–4413; b) A. Harada, R. Ko-
bayashi, Y. Takashima, A. Hashidzume, H, Yamaguchi, Nat. Chem.
2011, 3, 34–37; c) S. Tamesue, Y. Takashima, H. Yamaguchi, S. Shin-
kai, A. Harada, Angew. Chem. 2010, 122, 7623–7626; Angew. Chem.
Int. Ed. 2010, 49, 7461–7464; d) S. Dong, Y. Luo, X. Yan, B. Zheng,
X. Ding, Y. Yu, Z. Ma, Q. Zhao, F. Huang, Angew. Chem. 2011, 123,
1945–1949; Angew. Chem. Int. Ed. 2011, 50, 1905–1909; e) T. Aku-
tagawa, K. Kakiuchi, T. Hasegawa, S. Noro, T. Nakamura, H. Hase-
gawa, S. Mashiko, J. Becher, Angew. Chem. 2005, 117, 7449–7453;
Angew. Chem. Int. Ed. 2005, 44, 7283–7287; f) A. A. Sobczuk, S.
Tamaru, S. Shinkai, Chem. Commun. 2011, 47, 3093–3095; g) I.
Hwang, W. S. Jeon, H. J. Kim, D. Kim, H. Kim, N. Selvapalam, N.
Fujita, S. Shinkai, K. Kim, Angew. Chem. 2007, 119, 214–217;
Angew. Chem. Int. Ed. 2007, 46, 210–213; h) T. Becker, C. Y. Goh,
F. Jones, M. J. McIldowie, M. Mocerino, M. I. Ogden, Chem.
Commun. 2008, 3900–3902; i) X. Cai, K. Liu, J. Yan, H. Zhang, X.
Hou, Z. Liu, Y. Fang, Soft Matter 2012, 8, 3756–3761; j) J. Liu, P.
He, J. Yan, X. Fang, J. Peng, K. Liu, Y. Fang, Adv. Mater. 2008, 20,
2508–2511; k) X. Yan, D. Xu, X. Chi, J. Chen, S. Dong, X. Ding, Y.
Yu, F. Huang, Adv. Mater. 2012, 24, 362–369.
Study of enzyme immobilization and release from the supramolecular
gel: The gel loaded with FITC-CalB was prepared by dissolving FITC-
CalB (25 mL, 0.335 mgmLꢀ1 of the enzyme) and the gelator (3.15 mg) in
acetonitrile (250 mL) followed by sonication and gel formation as de-
ACHTUNGTRENNUNGscribed above. Then, in two experiments, either deionized water (750 mL)
or a solution of KPF6 (10 mg) in deionized water (750 mL) was added to
the gel sample. For both samples, the amount of released FITC-CalB was
followed by monitoring the change in fluorescence intensity (excitation
wavelength: 495 nm) at 518 nm, which corresponds to the fluorescence
maximum of FITC-CalB.
Immobilization and release of enzymes in/from Pickering emulsions: For
the immobilization of CalB in Pickering emulsions, the gelator (6.3 mg)
and an aqueous solution of CalB (50 mL, 0.335 mgmLꢀ1 of the enzyme)
were added to acetonitrile (450 mL). The enzyme/gelator mixture
(500 mL) was homogenized in dispersions of SiO2@TMODS nanoparticles
(silica nanoparticles 140 nm in diameter that are made hydrophobic with
trimethoxy(octadecyl)silane (TMODS)) in hexane (1.5 mL) at room tem-
perature. Jellified Pickering emulsions entrapping the enzyme were char-
acterized by confocal laser scanning microscopy (CLSM, Leica DM
IRBE confocal laser scanning microscope at an excitation wavelength of
488 nm). The CalB enzyme content was determined according to a Brad-
ford assay.[28] To examine the enzyme release from the Pickering emul-
sions, the same procedure was carried out as described for the enzyme re-
lease studies from the gel, except that the samples were centrifuged at
6000 rpm for 10 min to remove the hexane layer before the addition of
deionized water or KPF6 solution (750 mL), respectively.
Assessment of the catalytic performance of free and immobilized CalB:
CalB activity was determined by using the esterification of 1-octanol and
octanoic acid as the test reaction. Typically, the Pickering emulsion in
hexane (2 mL) containing different amounts of CalB were introduced
into hexane (13 mL) containing a calculated amount of substrate. For the
control experiment, CalB (50 mL) with or without acetonitrile (450 mL)
was introduced into a solution of the substrate in hexane (14.5 mL). The
overall water content was the same in all experiments. The esterification
reactions were all carried out under the same conditions with 100 mm 1-
octanol and 100 mm octanoic acid in hexane on a rotating shaker at
80 rpm at room temperature for 130 min. After 10, 20, 30, 40, and
130 min, aliquots (100 mL) were removed from the mixture and centri-
fuged at 14500 rpm for 20 s. Subsequently the supernatants were ana-
lyzed for ester concentration by gas chromatography (Shimadzu 2010;
BPX5 column from SGE: length 25 m, ID 0.22 mm; film thickness:
0.25 mm; detector: FID at 3008C; injector: 2758C, injection volume:
1 mL, split model; temperature program: start temperature 808C, hold for
0.5 min, temperature rise 208Cminꢀ1 from 80 to 1708C and 58Cminꢀ1
from 1708C to the end temperature 2008C). The concentrations of the
octyl octanoate product were calculated from the area of the chromato-
graphic peak at a retention time of 10.01 min. The catalytic activity was
assessed in terms of octyl octanoate units. One unit (U) correlates to the
production of one mmol octyl octanoate per minute. The specific activity
is expressed as units per milligram of protein (U mgꢀ1).
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Fibrous Networks Incorporating Macrocycles
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[6] a) J. H. Jung, H. Kobayashi, M. Masuda, T. Shimizu, S. Shinkai, J.
Am. Chem. Soc. 2001, 123, 8785–8789; b) H. Engelkamp, S. Middel-
beek, R. J. M. Nolte, Science 1999, 284, 785–788.
Dong, M. Zhang, X. Ding, Y. Yu, S. Shao, F. Huang, Chem.
Commun. 2011, 47, 7086–7088; e) W. Jiang, D. Sattler, K. Rissanen,
C. A. Schalley, Org. Lett. 2011, 13, 4502–4505.
[7] a) G. M. Whitesides, B. Grzybowski, Science 2002, 295, 2418–2421;
b) X. Yang, G. Zhang, D. Zhang, J. Mater. Chem. 2011, 21, 38–50;
c) Z.-X. Liu, Y. Feng, Z.-C. Yan, Y.-M. He, C.-Y. Liu, Q.-H. Fan,
Chem. Mater. 2012, 24, 3751–3757; d) M. D. Segarra-Maset, V. J.
Nebot, J. F. Miravet, B. Escuder, Chem. Soc. Rev. 2013, DOI:
10.1039/C2CS35436E.
[8] A. R. Hirst, B. Escuder, J. F. Miravet, D. K. Smith, Angew. Chem.
2008, 120, 8122–8139; Angew. Chem. Int. Ed. 2008, 47, 8002–8018.
[9] a) H. Komatsu, S. Matsumoto, S.-i. Tamaru, K. Kaneko, M. Ikeda, I.
Hamachi, J. Am. Chem. Soc. 2009, 131, 5580–5585; b) Z. Qi, P. Ma-
lo de Molina, W. Jiang, Q. Wang, K. Nowosinski, A. Schulz, M.
Gradzielski, C. A. Schalley, Chem. Sci. 2012, 3, 2073–2082.
[10] a) Y. Gao, F. Zhao, Q. Wang, Y. Zhang, B. Xu, Chem. Soc. Rev.
2010, 39, 3425–3433; b) A. E. David, A. J. Yang, N. S. Wang, Meth-
ods Mol. Biol. 2011, 679, 49–66.
[16] J. W. Steed, Chem. Soc. Rev. 2010, 39, 3686–3699.
[17] R. M. Versteegen, R. P. Sijbesma, E. W. Meijer, Macromolecules
2005, 38, 3176–3184.
[18] M.-O. M. Piepenbrock, G. O. Lloyd, N. Clarke, J. W. Steed, Chem.
Commun. 2008, 2644–2646.
[19] A. Lohr, M. Lysetska, F. Wꢂrthner, Angew. Chem. 2005, 117, 5199–
5202; Angew. Chem. Int. Ed. 2005, 44, 5071–5074.
[20] Calculations were performed with the CaChe 5.0 program package,
Fujitsu, Krakow/Poland.
[21] P. Jonkheijm, P. van der Schoot, A. P. H. J. Schenning, E. W. Meijer,
Science 2006, 313, 80–83.
[22] a) W. Jiang, H. D. F. Winkler, C. A. Schalley, J. Am. Chem. Soc.
2008, 130, 13852–13853; b) W. Jiang, C. A. Schalley, Proc. Natl.
Acad. Sci. USA 2009, 106, 10425–10429.
[23] a) P. Terech, S. Friol, Tetrahedron 2007, 63, 7366–7374; b) P. Lo Nos-
tro, R. Ramsch, E. Fratini, M. Lagi, F. Ridi, E. Carretti, M. Ambro-
si, B. W. Ninham, P. Baglioni, J. Phys. Chem. B 2007, 111, 11714–
11721; c) M. Burkhardt, S. Kinzel, M. Gradzielski, J. Colloid Inter-
face Sci. 2009, 331, 514–521.
[24] For a few selected examples, see: a) K. Martinek, A. Levashov, Y.
Khmelnitsky, N. Klyachko, I. Berezin, Science 1982, 218, 889–891;
b) C. Pizarro, M. A. Fernꢅndez-Torroba, C. Benito, J. M. Gonzꢅlez-
Sꢅiz, Biotechnol. Bioeng. 1997, 53, 497–506; c) Q. Wang, Z. Yang, Y.
Gao, W. Ge, L. Wang, B. Xu, Soft Matter 2008, 4, 550–553.
[25] a) M. B. Ansorge-Schumacher, Immobilization of biological cata-
lysts, Wiley-VCH, Weinheim, 2008; b) L. Cao, Carrier-bound Immo-
bilized Enzymes: Principles, Applications and Design, Wiley-VCH,
Weinheim, 2005; c) A. M. Klibanov, Science 1983, 219, 722–727;
d) W. Tischer, F. Wedekind, Top. Curr. Chem. 1999, 200, 95–126.
[26] a) M. B. Ansorge-Schumacher, Mini-Rev. Org. Chem. 2007, 4, 243–
245; b) G. Carrea, S. Riva, Angew. Chem. 2000, 112, 2312–2341;
Angew. Chem. Int. Ed. 2000, 39, 2226–2254; c) C. Wu, M. Kraume,
M. B. Ansorge-Schumacher, ChemCatChem 2011, 3, 1314–1319;
d) D. J. Cole-Hamilton, Science 2010, 327, 41–42.
[11] S. U. Pickering, J. Chem. Soc. 1907, 91, 2001–2021.
[12] a) C. Wu, S. Bai, M. B. Ansorge-Schumacher, D. Wang, Adv. Mater.
2011, 23, 5694–5699; b) Z. Wang, M. C. M. van Oers, F. P. J. T.
Rutjes, J. C. M. van Hest, Angew. Chem. 2012, 124, 10904–10908;
Angew. Chem. Int. Ed. 2012, 51, 10746–10750.
[13] a) J. H. van Esch, S. De Feyter, R. M. Kellogg, F. De Schryver, B. L.
Feringa, Chem. Eur. J. 1997, 3, 1238–1243; b) J. H. van Esch, F.
Schoonbeek, M. de Loos, H. Kooijman, A. L. Spek, R. M. Kellogg,
B. L. Feringa, Chem. Eur. J. 1999, 5, 937–950; c) F. S. Schoonbeek,
J. H. vanEsch, R. Hulst, R. M. Kellogg, B. L. Feringa, Chem. Eur. J.
2000, 6, 2633–2643; d) A. Arnaud, L. Bouteiller, Langmuir 2004, 20,
6858–6863; e) Y. Jeong, K. Hanabusa, H. Masunaga, I. Akiba, K.
Miyoshi, S. Sakurai, K. Sakurai, Langmuir 2005, 21, 586–594; f) L.
Bouteiller, O. Colombani, F. Lortie, P. Terech, J. Am. Chem. Soc.
2005, 127, 8893–8898; g) C. Wang, D. Zhang, D. Zhu, Langmuir
2007, 23, 1478–1482; h) M. Bellot, L. Bouteiller, Langmuir 2008, 24,
14176–14182.
[14] a) G. W. Gokel, W. M. Leevy, M. E. Weber, Chem. Rev. 2004, 104,
2723–2750; b) F. Huang, H. W. Gibson, Prog. Polym. Sci. 2005, 30,
982–1018; c) W. Jiang, C. A. Schalley, Beilstein J. Org. Chem. 2010,
6, 14.
[27] P. Terech, C. Rossat, F. Volino, J. Colloid Interface Sci. 2000, 227,
363–370.
[15] For examples of pseudo-rotaxanes formed with benzo-21-crown-7,
see: a) C. Zhang, S. Li, J. Zhang, K. Zhu, N. Li, F. Huang, Org. Lett.
2007, 9, 5553–5556; b) C. Zhang, K. Zhu, S. Li, J. Zhang, F. Wang,
M. Liu, N. Li, F. Huang, Tetrahedron Lett. 2008, 49, 6917–6920;
c) W. Jiang, A. Schꢃfer, P. C. Mohr, C. A. Schalley, J. Am. Chem.
Soc. 2010, 132, 2309–2320; d) X. Yan, M. Zhou, J. Chen, X. Chi, S.
[28] M. M. Bradford, Anal. Biochem. 1976, 72, 248–254.
Received: January 18, 2013
Revised: May 10, 2013
Published online: July 10, 2013
Chem. Eur. J. 2013, 19, 10150 – 10159
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
10159