Enzymatic hydrolysis-induced degradation of a lactose-coupled supramolecular hydrogel

Article abstract of DOI:10.1039/c8cc04727h

Amphiphilic urea 1 with a hydrophilic lactose group was prepared as a low-molecular-weight hydrogelator, which formed a transparent supramolecular hydrogel. Enzymatic hydrolysis of the lactose moiety using β-galactosidase allowed a gel-to-sol phase transition of the supramolecular hydrogel. A β-galactosidase inhibitor enables us to control the time course of this phase transition.

Full text of DOI:10.1039/c8cc04727h

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Enzymatic hydrolysis-induced degradation of a lactose-coupled
supramolecular hydrogel
Shuto Akama,a Takumi Makia and Masamichi Yamanaka*a,b
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Amphiphilic urea 1 with a hydrophilic lactose group was prepared disaccharidases, which catalyze the hydrolysis of
a
as a low-molecular-weight hydrogelator, which formed a disaccharide into two monosaccharides. Disaccharidases
transparent supramolecular hydrogel. Enzymatic hydrolysis of the located at small intestinal villus catalyze the degradation of
lactose moiety using β-galactosidase allowed the gel-to-sol phase supramolecular hydrogels to release the trapped drugs at the
transition of the supramolecular hydrogel. A β-galactosidase small intestine because of disaccharidases are localized at.23
inhibitor enables us to control the time course of this phase Among the different types of disaccharidases available, we are
transition.
interested in lactase (β-galactosidase: β
-Gal)24 because there is
a large difference in water solubility between the substrate
lactose (0.22 g/mL at 25 °C) and the product glucose (0.91
g/mL at 25 °C). LMWHG with a lactose hydrophilic group can
Hydrogels are attractive and useful materials that have found
widespread application found in our modern society, e.g. soft
contact lens, paper diaper, cosmetics, stationery, etc.
Hydrogels also play an important role in advanced medicine
and biotechnology research, such as drug delivery systems
(DDS), regenerative medicine, cell culture, etc.1 Control of
the gelation process via an in situ enzymatic reaction has great
advantages for biological applications. Enzyme-responsive
polymer hydrogels have been reported, mainly based on the
decomposition or construction of crosslinking points via
enzymatic reactions.8-10 In supramolecular hydrogels,11-13 the
formation of which is driven by non-covalent self-assembly, it
is possible to confer more diverse enzyme responsiveness. 14,15
Supramolecular hydrogels can be formed spontaneously by
linking the two precursors via enzymatic reaction and in situ
be hydrolyzed by
β-Gal to produce an amphiphile with a
glucose hydrophilic group, and this change in hydrophilicity
induces a gel-to-sol phase transition. In this paper, we report
the synthesis and gelation of a lactose-coupled amphiphilic
urea, and the enzymatic hydrolysis-induced gel-to-sol phase
transition of the supramolecular hydrogel.
7
–
Sugar-derived amphiphilic compounds are a common
structure among LMWHGs.25-30 Based on these pioneering
studies and our previous research on tris-urea gelators,31 we
designed an amphiphilic urea
1 bearing a hydrophilic lactose
group as a novel LMWHG, synthesized in only two steps from
commercial compounds (Scheme 1). The key step of the
synthesis strategy is the aminoglycosylation reaction between
the amino group of the hydrophobic part and the hydrophilic
unprotected sugar to construct the amphiphilic structure.32
Protection and de-protection of the sugar hydroxyl groups can
be avoided in this strategy, which makes the strategy
advantageous in terms of step economy. Reaction of 1,4-
phenylenediamine with cyclohexyl isocyanate in 1:1 molar
ratio gave the unsymmetrical N-p-aminophenyl-N’-cyclohexyl
synthesis
of
a
low-molecular-weight
hydrogelator
(LMWHG).16,17 Enzymatic transformation, such as hydrolysis of
precursors to LMWHGs, also realized spontaneous formation
of supramolecular hydrogels.18-20 Some types of
supramolecular hydrogels underwent degradation by
enzymatic conversion of LMWHGs to non-gelling
molecules.21,22
For the application of supramolecular hydrogels as a drug
carrier, it is preferable to control its degradation by an enzyme
present at a particular site. For this purpose, we focused on
urea
and unprotected lactose in the presence of a catalytic amount
of ammonium sulfate afforded the desired amphiphilic urea
in 89% yield. Thus, synthesized over two steps without
column purification. The structure of
was confirmed by 1H
and 13C NMR and mass spectroscopy. The aminoglycosylation
2 in 88% yield. Aminoglycosylation reaction between 2
1
1
1
reaction occurred stereoselectively to yield only the
of the desired product.33
β-isomer
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Amphiphilic urea
1
acts as an LMWHG, and supramolecular using a rheometer. The storage moduli (G′) of the hydrogel of
in water with gentle were approximately 12-times higher than the loss moduli
heating followed by cooling to room temperature. The (G′′). Both types of moduli were almost independent of the
minimum gelation concentration (MGC) of in this gelation frequency in the range of 0.01 to 10 Hz (Fig. S1, ESI). The G′
method was estimated to be 0.3 wt% (5.4 mM) (Fig. 1). The and G′′ values of the 0.5 wt% supramolecular hydrogel of are
hydrogels were obtained by dissolving
1
1
1
1
resulting supramolecular hydrogels are highly transparent, and 19.6 and 1.7 kPa, respectively, at 1.0 Hz. The G′ and G′′ values
their transparencies were evaluated from their transmittance improved to 129 and 10.2 kPa, respectively, when the
spectra (Fig. 2). The non-gelated aqueous solution of
wt%) is clear with a transmittance of 100% over the entire supramolecular hydrogels of
visible region. High transparency, similar to that of the response typical of supramolecular hydrogels (Fig. S1, ESI).
solution, was observed for the supramolecular hydrogel of at
low concentration (0.3 wt%). Transmittance of over 90% was
found in supramolecular hydrogel obtained using 0.5 wt% of
The transparencies of the supramolecular hydrogels decreased
further upon increasing the concentration of to 0.75 wt%
1
(0.1 concentration of
1
was 1.0 wt%. Strain sweeps of the
1
demonstrate an elastic
1
1.
1
and 1.0 wt%. The scanning electron microscopy (SEM) image
of the xerogel showed fibrous aggregates with diameters
ranging from 200 to 700 nm (Fig. 1).
H2N
H2N
O
+
N
N
OCN
OH
NH2
CH2Cl2, rt
OH
H
H
88%
2
HO
O
Lactose
(NH4)2SO4
O
OH
1
HO
O
HO
H
OH
N
O
MeOH, ꢀ
89%
Scheme 1. Synthesis of amphiphilic urea 1
N
N
Fig.
2 Transmittance spectra of aqueous mixtures of 1 with the following
H
H
concentrations: (a) 0.1 wt% (sol); (b) 0.3 wt% (gel); (c) 0.5 wt% (gel); (d) 0.75 wt% (gel);
(e) 1.0 wt% (gel).
An interesting property of
hydrogels could be prepared without heating simply by stirring
a mixture of and water using a vortex mixer (2500 rpm) for 5
1 is that supramolecular
1
min at room temperature. In this method, the MGC was 0.5
wt%, and it was impossible to avoid the presence of trace
amounts of dissolved solids
hydrogel could also be prepared by injecting a DMSO solution
of into pure water stirred with a magnetic stirrer (Fig. S2,
ESI).
Next, the
supramolecular hydrogel of
solution of -Gal (100 units) was added to the top surface of
the supramolecular hydrogel of (0.5 wt%, 500 L) and its
1 (Fig. S2, ESI). The supramolecular
1
β
-Gal responsive macroscopic alteration of
was investigated. An aqueous
1
β
1
ꢁ
time course was observed (Fig. 3). The supramolecular
hydrogel underwent gradual gel-to-sol phase transition from
the surface in contact with
β-Gal, and disintegrated into a
Fig. 1 Photograph of supramolecular hydrogels of 1 (0.3 wt%) (inset) and SEM image of
suspension after 6 h. The SEM image of the suspended mixture
still showed fibrous aggregates (Fig. S3, ESI). The time required
for the complete phase transition increased with decreasing
the xerogel.
A typical characteristic of supramolecular hydrogels is
34
increasing thermal stability (Tgel
increasing the concentration of
amount of the added
(0.5 wt%, 500 L) completely disintegrated into a suspension
after 9 h in the presence of 10 units of -Gal (Fig. S4, ESI).
The -Gal-induced gel-to-sol phase transition was thought
to occur due to the hydrolytic structural transformation of the
lactose moiety of . An amphiphilic urea , which is an
expected product of the -Gal-induced hydrolysis of , was
β-Gal; the supramolecular hydrogel of 1
)
of the hydrogel upon
ꢁ
1
(Tgel; 67 °C for 0.5 wt%, and
β
76 °C for 1.0 wt%). These hydrogels exhibited thermo-
responsive behavior, and reversible gel-to-sol phase
transitions occurred upon heating and cooling the mixtures.
β
1
3
The physical properties of the supramolecular hydrogels of
1
β
1
were evaluated by measuring their viscoelastic properties
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synthesized by aminoglycosylation between
2
and D-glucose. of PETG used (0.1 mol% to
1
) is not sufficient to hinder the
The compound
image of the precipitate showed fibrous aggregates (Fig. S5, required for the complete phase transition was extended to 8
ESI). These results support the fact that the -Gal-induced gel- h upon using a supramolecular hydrogel of prepared from a
to-sol phase transition of the supramolecular hydrogel of 0.1 mM PETG solution (500 L). The time required for the
occurs due to the formation of by hydrolysis of complete phase transition was further extended to 2 days
Furthermore, ¹H-NMR analysis showed that the amphiphilic upon using 1.0 mM PETG solution. When 10 units of -Gal was
is a major component of the suspension resulting from used, the time required for the phase transition of
-Gal-induced decomposition of the supramolecular supramolecular hydrogel of prepared from PETG solution
(Fig. 3 and Fig. S6, ESI). could be extended. The time required for completion of the
phase transition of the supramolecular hydrogel of 0.5 wt%
3 formed a suspension in water, and the SEM phase transition. Keeping all other variables constant, the time
β
1
1
ꢁ
3
1.
β
urea
the
3
β
1
hydrogel of
1
1
to the sol was 9 h, 1 day, 2 days, and 3 days when the PETG
concentration was 0, 0.01, 0.1, and 1.0 mM, respectively.
Table 1 Time required for phase transition supramolecular hydrogels of 1a
PETG (mM)
β-Gal (units)b
0.0
6 h
9 h
0.01
6 h
0.1
8 h
2 d
1.0
2 d
3 d
100
10
1 d
a) Supramolecular hydrogels of 1 (0.5 wt%) were prepared from 500 ꢁL of PETG
solutions. b) 50 ꢁL of β-Gal solutions were placed on the top surfaces of
supramolecular hydrogels of 1.
The
humans, and in plants; for instance, kiwifruit contains plenty of
-Gal.36 Therefore, when
kiwifruit was attached to
supramolecular hydrogel of , the gel-to-sol phase transition
progressed in a manner similar to when pure -Gal was
β-Gal enzyme is widely present in animals, such as
β
a
1
β
applied. Supramolecular hydrogel (0.5 wt%, 10 mL) was
prepared and a slice of kiwifruit (1.5 g) was placed on its top
surface. The gel-to-sol phase transition proceeded from the
interface in contact with the kiwifruit (Fig. 4). It took 24 h for
the complete phase transition, and a clear solution was
Fig. 3 (A) Photographs of the time course of a mixture of supramolecular hydrogel (0.5
wt% of 1, 500 ꢁL) and β-Gal (100 units, 50 ꢁL). (B) 1H NMR spectra (DMSO-d6, 298 K) of
a) 1; b) 3; c) mixture of supramolecular hydrogel (0.5 wt% of 1, 500 ꢁL) and β-Gal (100
units) after 6 h; d) mixture of supramolecular hydrogel (0.5 wt% of 1, 10 mL) and
kiwifruit (1.5 g) after 24 h.
obtained after the phase transition unlike when pure
was used. The 1H-NMR spectrum of this solution was more
complicated than that of the product of the phase transition
β-Gal
OH
OH
HO
HO
O
HO
HO
H
N
O
using pure
β
hydrolysis of the lactose moiety of
-Gal (Fig. 3 and Fig. S6, ESI). In addition to the
, other degradation
O
OH
S
Ph
1
OH
N
N
3
H
H
PETG
reactions have been mediated by enzymes contained in raw
kiwifruit.
Chart Chemical structures of 3 and PETG
In order to use supramolecular hydrogel as a substrate for
controlled release of drugs, the time required for the gel-to-sol
phase transition should be controlled by varying factors other
than enzyme concentration. We proposed that the phase
transition time could be controlled by mixing an inhibitor of
the enzyme with the supramolecular hydrogel. 2-Phenylethyl
Fig. 4 Photographs of the time course of a mixture of supramolecular hydrogel (0.5 wt%
of 1, 10 mL) and kiwifruit (1.5 g).
β
-D-thiogalactoside (PETG), which associates strongly with
Gal (association constant at 25
C = 1.0 × 106 M−1), has been
used as an inhibitor of
-Gal.35 The
transition of supramolecular hydrogel of
β-
º
β
β
1
-Gal-induced phase
could be delayed in
In this study, we developed an amphiphilic urea
1 with
lactose as a hydrophilic group as an LMWHG, in only two steps
from commercial compounds without column chromatography
purification. Highly transparent supramolecular hydrogel was
the presence of PETG (Table 1). When 100 units of
added to a 0.5 wt% supramolecular hydrogel of
from an aqueous solution of PETG (0.01 mM, 500
β
-Gal was
prepared
L), it
1
ꢁ
formed (MGC of
1
= 0.3 wt%), which underwent gel-to-sol
-Gal, an enzyme for lactose
required 6 h for the complete gel-to-sol phase transition. This
phase transition upon adding β
hydrolysis. The time required for this phase transition could be
result is the same as that obtained using supramolecular
hydrogel of
1 prepared from pure water, because the amount
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Manuscript ID: CC-COM-06-2018-004727
Title: Enzymatic hydrolysis-induced degradation of a lactose-coupled supramolecular hydrogel
Authors: Shuto Akama, Takumi Maki and Masamichi Yamanaka
A lactose-coupled amphiphilic urea self-assembled to form supramolecular hydrogel, which
responsive enzymatic hydrolysis.
1