Enzymatically derived sugar-containing self-assembled organogels with nanostructured morphologies

Article abstract of DOI:10.1002/anie.200600989

The long and short of it: A regioselective enzyme-catalyzed acylation of the disaccharide trehalose generated a family of low-molecular-weight gelators with unprecedented properties. The selectivity of enzymatic catalysis enables direct control over gelation properties by simply varying the acyl-chain length to give gelation in solvents ranging from the highly hydrophilic acetonitrile to the highly hydrophobic cyclohexane. (Chemical Equation Presented).

Full text of DOI:10.1002/anie.200600989

Communications
Organogels
To that end, we examined the synthesis of sugar-based
diesters by using the lipase B from Candida antarctica
(CALB). Transesterification reactions were performed in
acetone that contained either vinyl stearate or vinyl butyrate
as highly or moderately hydrophobic ester donors, respec-
tively, and with several common disaccharides including
sucrose, maltose, lactose, and trehalose. Interestingly, only
the reactions with trehalose, a symmetrical disaccharide with
an a-1,1 glycosidic bond, resulted in gel formation during the
course of the transesterification reactions (Table 1, gelators 2
and 5), thereby confirming the importance of monomer
structure in gel assembly. Trehalose-6,6’-distearate and tre-
halose-6,6’-dibutyrate were obtained as the sole products
from the respective enzymatic reactions in yields of > 50%.
Hence, CALB was highly regiospecific in its acylation of
trehalose. The purified dieters were tested in a wide range of
solvents for their gelation ability (see the Supporting Infor-
mation). The trehalose distearate was insoluble in water and
soluble in chloroform and 1,4-dioxane, whereas trehalose 6,6’-
dibutyrate was insoluble in cyclohexane and olive oil and
soluble in water. Gels were formed in all other solvents tested.
As a result of these studies, we generated a series of
additional diesters with chain lengths of C2 to C14 (1, 3, 4, and
6) and assessed their gelation capacity in several key solvents,
ranging from the hydrophilic acetonitrile to the hydrophobic
DOI: 10.1002/anie.200600989
Enzymatically Derived Sugar-Containing Self-
Assembled Organogels with Nanostructured
Morphologies**
George John, Guangyu Zhu, Jun Li, and
Jonathan S. Dordick*
Control of building-block assembly and phase behavior is
crucial for the ultimate design of functional architectures
ranging from the nano- to the macroscales, with organogels
representing one important example of this functional
architecture. An ideal gelator is an amphiphile that induces
gel formation through self-assembly into highly ordered
structures in which the hydrophilic moieties interact through
extensive hydrogen bonding and the hydrophobic moieties
interact with the organic liquid. Among the growing list of low
molecular weight compounds that induce gelation, sugars
appear to satisfy these requirements, and numerous alkyl- and
aryl-based monosaccharide gelators have been generated.
As opposed to chemical synthesis, enzymatic catalysis is
highly selective and has been used to generate low-molecular-
weight compounds that can gel organic solvents, generate a
[
1]
[
2]
[
3]
p-xylene (Table 1). The minimum gelator concentration (cmin
)
[
4]
required to induce gelation is strongly dependent on the acyl-
chain length. In most of the cases, a shorter chain length
promotes gelation at lower gelator concentration, with the
exception of gelation in acetonitrile and isopropanol. These
results sharply contrast with typical sugar-based amphiphilic
organogels, which require long-chain alkyl or aryl moieties to
[
5]
wide range of sugar-based esters, and in particular, prepare
[
6]
highly regioselective symmetrical diesters. We reasoned that
such a biocatalytic approach would provide an alternative
route to the synthesis of disaccharide-based diesters with
physical and structural features appropriate for a low-
molecular-weight gelator along with a controlled symmetry
that may aid in self-assembly. Combining the principles of
supramolecular chemistry with the selectivity of biocatalysis
may represent a new and powerful strategy to develop new
molecularly defined and functional materials.
[
3a–e]
induce gelation.
Surprisingly, the trehalose-6,6’-diacetate
(1) was capable of inducing gelation at a cmin of 0.04% (w/v;
0.84 mm) in ethyl acetate and nearly this low in methyl
methacrylate. This represents, to our knowledge, the lowest
[
7]
cmin value reported for a sugar ester gelator. For ethyl
acetate, the cmin represents over 12000 solvent molecules
being associated per molecule of 1. This can be translated into
a swelling of the weight of the gelator approximately 2500-
fold.
[
*] Dr. G. Zhu, Prof. J. S. Dordick
Department of Chemical and Biological Engineering and
Rensselaer Nanotechnology Center
Rensselaer Polytechnic Institute
Troy, NY 12180 (USA)
The trehalose diesters are excellent gelators over a broad
range of organic solvents (see the Supporting Information)
and in a mixture of solvents. For example, in the case of a 1:1
binary mixture of ethyl acetate and acetonitrile, the minimum
gelation concentration was between the cmin values in the pure
solvents (see the Supporting Information), therefore showing
no preference for a given solvent. Interestingly, the longer-
ester-chain trehalose derivatives could form gels in olive oil
(Table 1) with relatively low cmin values. Addition of 5% free
oleic acid, which would be present in low-purity olive oil, did
not affect the swelling capacity of the gel. Hence, complex,
multicomponent solvent systems, such as that found in a
natural oil and in the presence of a charged hydrolysis
product, could be subject to gelation.
Fax: (+1)518-276-2207
E-mail: dordick@rpi.edu
Prof. G. John
Department of Chemistry
The City College of the City University of New York
New York, NY 10031 (USA)
Dr. J. Li
Department of Polymer Science
The University of Southern Mississippi
Hattiesburg, MS 39406 (USA)
[
**] We thank Dr. Maura Weathers of the Cornell Center for Material
Research (CCMR) for small-angle X-ray scattering (SAXS) meas-
urements and Dr. Praveen of CCNY, CUNY for calculations. This
research was supported by an NSF-Nanoscale Science and
Engineering Center at Rensselaer (DMR-0117792).
As described above, although regiospecificity was
achieved to give the respective 6,6’-diesters for all disacchar-
ides tested, gel formation only occurred with trehalose,
suggesting that the unique symmetry of this molecule, which
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
4
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Angew. Chem. Int. Ed. 2006, 45, 4772 –4775

Angewandte
Chemie
Table 1: Minimum gelation concentration (weight percent) of trehalose-based diesters in different
solvents at 258C.
xerogels of 1 in ethyl acetate (Fig-
[
a]
ure 1a,b) and in isopropanol (Fig-
ure 1c,d), as well as 6 in ethyl
acetate (Figure 1e,f). These xero-
gels consist of 3D entangled fiber-
like aggregates with diameters of
1
0–500 nm and lengths in the
micron scale. The high aspect
ratios of the gel fibers clearly
indicate that the intergelator inter-
actions are highly anisotropic.
Most likely, the fibers observed in
the electron micrographs consist of
bundles of gelator aggregates, sim-
ilar to the structures observed in
other gel systems. The solvent
clearly influences the gel structure,
for example, the ethyl acetate gel
of 1 is transparent and shows
extended fibrous structures (Fig-
ure 1a), whereas the isopropanol
gel of 1 is opaque and consist of
larger and more crystalline fibers
(Figure 1c). Furthermore, the fact
Solvent
R=
1
-CH3
2
3
4
5
6
(
log P
-(CH ) CH -(CH ) CH -(CH ) CH -(CH ) CH -CH=CH2
2 2 2 8 3 2 12 3 2 16 3
value)
acetonitrile
G (0.36) G (0.69)
G (0.34) G (1.0)
G (0.54) G (1.39)
G (0.04) G (0.13)
G (0.05) G (0.11)
G (0.18)
G (1.3)
G (0.11)
G (1.4)
G
G
G
G
(
-0.34)
Acetone
-0.24)
isopropanol
0.05)
ethyl acetate
0.73)
methyl methacrylate
1.38)
p-xylene
3.15)
olive oil
N/A)
G
(
G (2.21)
G (0.71)
G (0.82)
G (0.18)
G (0.09)
G (1.31)
G (1.1)
G
(
G (0.72)
G (0.40)
G (0.37)
G (0.18)
G
(
G (0.85)
G (0.25)
G (0.13)
G
(
I
I
G (0.14)
I
G (0.72)
I
(
(
that low gelator concentrations can
[a] G indicates that the gel formed and I indicates that the gelator could not dissolve in the organic
yield gels with the weak hydro-
solvent at elevated temperatures.
phobicity of the C2 acyl moiety
suggests that H-bonding is the
was maintained by the regioselectivity of lipase-catalyzed
acylation, may play the key role in its strong gelation ability.
To test this hypothesis, we chemically acylated trehalose with
stearoyl chloride to yield a mixture of trehalose esters.
Following isolation of the diesters (a mixture of regioisomers
among the eight free hydroxy groups, data not shown),
gelation studies were performed in ethyl acetate. Gelation did
not occur until a diester mixture concentration of approx-
imately 10% (w/v) was reached; well over 10-fold higher than
that required by the 6,6’-distearate. When the 6,6’-distearate
was purified from the diester mixture, identical gelation
properties to those synthesized enzymatically were obtained.
Similar results were obtained with the 6,6’-diacetate and 6,6’-
dibutyrate derivatives. These results indicate that gelation is
strongly favored with highly symmetrical disaccharide ester
derivatives that can be synthesized through regioselective
enzymatic catalysis.
predominant mechanism for gel assembly, although hydro-
phobic interactions may also contribute to gelation at longer
acyl-chain lengths. The ability of H-bonding to dominate gel
assembly is supported by the extremely high hydroxy-group
density in a disaccharide like trehalose, as depicted in
Figure 2e (for more detail, see the Supporting Information).
Differential scanning calorimetry (DSC) of gels 1–4 was
performed in ethyl acetate to yield a gel!sol transition
temperature as a function of gelator mole fraction and
therefore enable calculation of gel melting enthalpy (DHm;
see the Supporting Information). Values of DH for gelators
m
1
–4 were determined to be approximately 55, 44, 30, and
ꢀ
1
2
2 kJmol , respectively. The high DH of 1 indicates that this
m
gelator is the most effective in forming highly stable gels in
ethyl acetate, which is consistent with its low cmin. Longer acyl
chains favor greater solvation of the diester in ethyl acetate
and therefore reduce their ability to form strong gels.
The morphological properties of the trehalose-based
organogels were obtained through scanning electron micro-
scopy (SEM). Figure 1 depicts selected SEM images of the
Figure 1. FE-SEM images of the organogels from a) 1 in ethyl acetate,
b) 1 in ethyl acetate at a higher magnification, c) 1 in isopropanol, d) 1
in isopropanol at a higher magnification, e) 6 in ethyl acetate; inset
shows higher magnification of 6 in ethyl acetate, f) 6 self-supporting
and porous scaffold after UV polymerization in ethyl acetate.
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Communications
Figure 2. Proposed scheme of molecular packing. a) Gel formed by 1 in ethyl acetate, b) 3D network, c) fibers, d) multilayers, and e) modeled
molecular packing. f) SAXS data for ethyl acetate gel of 1.
To gain additional insight into the structures that comprise
the gel formed from 1, small-angle X-ray diffraction (XRD)
was employed. XRD of the wet gel gave a weak Bragg
reflection at 1.56 nm, indicating that the sugar diester
assembled into a well-ordered structure (Figure 2 f). This
reflection is approximately the same as that of the molecular
length of 1, which was confirmed by crystalline-sample
measurements (1.25 nm) and molecular modeling studies by
using energy-minimized calculations. XRD measurements
of the gels from 2–6 also gave similar diffraction patterns
showing that, in a suitable organic solvent, the trehalose
diesters self-assemble into ordered structures. Based on these
results, we propose a molecular arrangement of the trehalose
diesters in the organic liquid (Figure 2e). Molecular stacking
of the multilayers (Figure 2d) leads to the formation of gel
fibers (Figure 2c) followed by further growth into fibrous 3D
networks (Figure 2b) and finally formation of the gel
in the presence of a small amount of added water. Indeed
ꢀ1
CALB (0.5 mgmL ) in the presence of 2% (v/v) water
caused the ethyl acetate gel of 1 to undergo disintegration
with concomitant formation of free trehalose and some
trehalose 6-acetate.
Gels containing acrylate esters (for example, 6) can be
[
11]
further subjected to post-gelation cross-linking
in the
presence of 2,2-dimethoxy-2-phenylacetophenone (5 mol%)
as the photoinitiator and subsequent polymerization through
UV irradiation. Following solvent evaporation, the cross-
linked organogel from 6 was lyophilized to yield a highly
porous material (Figure 1 f). This material has a far-larger
pore structure than the gel from 6, which is not cross-linked
(Figure 1e). This suggests that it is suitable as a porous
scaffold. No gel shrinkage occurred during lyophilization.
Following drying into the aerogel, the cross-linked material
remained intact as a self-supporting scaffold (Figure 3a). This
material was capable of behaving as a modest hydrogel;
within 5 h the gel absorbed its weight 12-fold in water to give a
self-supporting transparent material (Figure 3b). We believe
that this is the first example of the generation of nano-
structures from self-assembled precursors in organic solvents
with hydrogel function.
[
8]
(
Figure 2a). The transparency of the resulting ethyl acetate
gel of 1 attests to a low-gelator-volume fraction along with a
nanoscale fiber size that does not interfere with light trans-
mission. Additional information on the packing arrangement
of the trehalose-6,6’-distearate (5) gelators through H-bond-
1
ing was further obtained by temperature-dependent H NMR
spectroscopic measurements in isopropanol and acetone (see
the Supporting Information). The gelators exhibit peaks in
both the sol and gel states, and as expected, the peak width
becomes sharper when the temperature increases above the
Tgel (468C). The peak width at 2.17–2.21 ppm (COOCH2
adjacent to the sugar) decreases below Tgel but remains
In conclusion, we have used a biocatalytic strategy to
design and synthesize a novel family of highly symmetrical
trehalose diesters that self-assemble in a range of organic
solvents and form gels at concentrations as low as 0.04% (w/v).
The gel fibers, particularly those obtained from short acyl-
chain length, are self-assembled and stabilized most likely
through the extensive H-bonding networks that are available
in the sugar; although both van der Waals packing and
hydrophobic interactions are also expected to contribute to
gel stability particularly as the acyl-chain length increases.
Combining the principles of supramolecular chemistry and
the selectivity of biocatalysis represents a powerful strategy to
develop new molecularly defined and functional materials.
The organogels reported herein may find potential applica-
tions in the food, pharmaceutical, and cosmetic industries in
constant at T> T . This result is consistent with the loosening
gel
of H-bonds that occur at the gel!sol transition temperature.
1
Interestingly, the organogels retain visible H NMR spectro-
scopic peaks in the gel state, suggesting that the gelator
[
3d,e,9]
molecules maintain sufficient thermal motion
to other gelating systems.
in contrast
[
10]
Although the gel structure can be dissociated in several
ways, such as by adding a good solvent of the gelator, another
route to degradation of these particular trehalose diester gels
is through selective ester-bond hydrolysis catalyzed by lipase
[
12]
which trehalose is already used routinely. In particular, the
4
774
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Angew. Chem. Int. Ed. 2006, 45, 4772 –4775

Angewandte
Chemie
Gel!sol transition temperatures (T ) were determined by DSC
m
with a Mettler DSC-822 differential scanning colorimeter equipped
with a nitrogen-gas cooling system. Field emission (FE)-SEM
measurements were carried out with a JEOL electron microscope.
A piece of the gel was placed on a carbon-coated copper grid and
dried for 3 h under vacuum before imaging. XRD measurements were
conducted by using a Bruker axs-D8 Discover with GADDS
diffractometer with graded d-space elliptical side-by-side multiplayer
optics, monochromated CuKa radiation (40 kV, 40 mA), and imaging
plate. The organogel was used as prepared in wet conditions for the
analysis. The typical exposure time was 1 min for self-assembled
structures with a 100 mm camera length.
Received: March 13, 2006
Published online: June 8, 2006
Keywords: enzyme catalysis · hydrogen bonds · organogels ·
.
self-assembly · trehalose
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6
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Experimental Section
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4
to remove the solid enzyme. The crude products were purified by
flash chromatography by using an ethyl acetate/methanol/water
(
17:4:1 v/v) mixture as the eluent. The yields of the isolated products
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ranged from 50–80%. The trehalose diesters were analyzed by
standard spectroscopic and elemental analysis procedures.
Angew. Chem. Int. Ed. 2006, 45, 4772 –4775
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
4775