MCM-enzyme-supramolecular hydrogel hybrid as a fluorescence sensing material for polyanions of biological significance.

Article abstract of DOI:10.1021/ja900500j

Polyanions are important sensing targets because of their wide variety of biological activities. We report a novel polyanion-selective fluorescence sensing system composed of a hybrid material of supramolecular hydrogel, enzymes, and aminoethyl-modified M

Full text of DOI:10.1021/ja900500j

Published on Web 03/24/2009
MCM-Enzyme-Supramolecular Hydrogel Hybrid as a
Fluorescence Sensing Material for Polyanions of Biological
Significance
Atsuhiko Wada,† Shun-ichi Tamaru,† Masato Ikeda,† and Itaru Hamachi†,‡,
*
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering,
Kyoto UniVersity, Katsura, Kyoto, 615-8510, Japan, and Japan Science and Technology Agency
(JST), CREST, 5 Sanbancho, Chiyoda-ku, Tokyo, 102-0075, Japan
Received January 22, 2009; E-mail: ihamachi@sbchem.kyoto-u.ac.jp
Abstract: Polyanions are important sensing targets because of their wide variety of biological activities.
We report a novel polyanion-selective fluorescence sensing system composed of a hybrid material of
supramolecular hydrogel, enzymes, and aminoethyl-modified MCM41-type mesoporous silica particles
(NH2-MCM41) encapsulating anionic fluorescent dyes. The rational combination of the polyanion-exchange
ability of NH2-MCM41 and semi-wet supramolecular hydrogel matrix successfully produced three distinct
domains; namely, cationic nanopores, hydrophobic nano/microfibers, and aqueous bulk gel phase, which
are orthogonal to each other. The coupling of anion-selective probe release from NH2-MCM41 with
translocation of the probe facilitated by enzymatic reaction enabled fluorescence resonance energy transfer-
type sensing in the hybrid materials for polyanions such as heparin, chondroitin sulfate, sucrose octasulfate,
and so forth. The enzymatic dephosphorylation catalyzed by phosphatase (alkaline phosphatase or acid
phosphatase) that is embedded in gel matrix with retention of activity also contributed to improving the
sensing selectivity toward polysulfates relative to polyphosphates. It is clear that the orthogonal domain
formation of these materials and maintaining the mobility of the fluorescent dyes between the three domains
are crucial for the rapid and convenient sensing provided by this system.
Introduction
ATP is a well-known main energy source for the majority of
cellular functions, and IP3 is one of the important secondary
Polyanions such as polysulfates, including heparin, chon-
droitin sulfate, and sucrose octasulfate (Suc-8S), and polyphos-
phates, including adenosine triphosphate (ATP), 1,4,5-inositol
triphosphate (IP3), inositol hexaphosphate (IP6), phosphorylated
proteins, polynucleotides such as DNA and RNA, and phos-
pholipids, are ubiquitous in nature. They all express a variety
of biological activities. For instance, heparin, a highly sulfated
glycosaminoglycan, is commonly used as an injectable antico-
agulant,1 and chondroitin sulfate, which is another sulfated
glycosaminoglycan and is found in cartilage, is widely used as
a dietary supplement to treat arthritis.2 Suc-8S has been recently
reported to stimulate fibroblast growth factor (FGF) signaling
through increasing FGF-FGF receptor affinity and promoting
receptor dimerization, which results in wound healing by
enhancing FGF-induced angiogenesis, as in the case of heparin
and heparin sulfate proteoglycans.3 Among polyphosphates,
messengers of signal transduction in live cells.4 IP6, which is
abundant in many plant sources, has recently received much
attention as nutrition against phosphate crisis5a-c and for its role
in cancer prevention and control of tumor growth, progression,
and metastasis.5d,e Thus, the development of rapid, convenient,
and high-throughput sensing systems for such polyanions is of
considerable significance. In particular, contamination by over-
sulfated chondroitin sulfate found in certain lots of heparin was
recently reported to cause serious side effects presumably due
to anaphylactoid response.6 Simple and selective detection of
such polysulfates is now in much demand; however, polysulfate-
selective sensors have been poorly explored compared with
sensors for polyphosphates.7,8
(4) Irvine, R. F. FEBS Lett. 1990, 263, 5–9.
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† Kyoto University.
‡ JST.
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Fromell, G. J.; Goodman, S.; Langer, A.; Califf, R. M.; Fox, K. A. A.;
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A R T I C L E S
Wada et al.
As an example of molecular sensors for polyanions operating
in solution, Anslyn’s group recently succeeded in developing
artificial receptors with phenylboronic acid and ammonium
group, which can bind anionic polysaccharide such as heparin
through boronate ester formation and electrostatic interaction.9
Although these molecular receptors are powerful,10 this approach
often suffers from the tedious synthetic procedures and complex
design required, especially for complicated sensing targets. As
a unique alternative, Matile’s group exploited fluorogenic sensor
systems comprising molecular assemblies such as vesicles
functionalized with engineered rigid-rod ꢀ barrels as the
nanopore that is selectively blocked by an analyte of interest.11
They demonstrated that the difference in the pore blockage
efficiency of polyanions such as ATP and ADP can be
monitored as the fluorogenic release of self-quenched fluorescent
dye. Very recently, Amoro´s and co-workers reported that
mesoporous silica particles (MCM41) bearing well-ordered
nanopores, the surface of which was functionalized to be
cationic, can be also utilized as colorimetric sensors on the basis
of ion-exchange phenomena between encapsulated anionic dyes
and simple anions such as carboxylates and phosphates.12,13
In contrast to these solution-based analyses, array technology
immobilizing sensing devices on solid supports is now rapidly
growing to afford DNA/protein/peptide/saccharide arrays for
high-throughput and efficient analysis.14 We recently proposed
a chemosensor array in which supramolecular hydrogels were
employed as a semi-wet matrix for noncovalently immobilizing
a variety of chemosensors.15,16 Here, we describe a fluorescence
sensing system consisting of a hybrid material of supramolecular
hydrogel, enzyme, and MCM41-type mesoporous silica particles
encapsulating a fluorescent probe in the pores. In this hybrid
system, three distinct microdomains, namely the cationic
nanopores, hydrophobic nano/microfibers, and aqueous gel bulk
phase, were produced in semi-wet condition and are orthogonal
to each other. Coupling of the anion-selective release of the
probe from NH2-MCM41 with translocation facilitated by
enzymatic reaction successfully yielded a unique fluorescence
resonance energy transfer (FRET)-type sensing material for
polyanions such as heparin, Suc-8S, and so forth. Dephospho-
rylation catalyzed by enzymes embedded in the gel phase
improved the discrimination ability of the sensor system toward
polysulfates relative to polyphosphates. The orthogonal domain
formation and sufficient mobility of the embedded molecules
are crucial for such a cooperative sensing system. We also
constructed an MCM41-hybrid array capable of distinguishing
polyanions such as heparin, Suc-8S, and IP6 with high throughput.
Results and Discussion
Polyanion Responsive Release of Fluorescent Probe
Entrapped within Amine-Modified MCM41. It was reported that
the interior of MCM41 is able to entrap anionic species by
appropriate modification with cationic amines.12,13,17 We pre-
pared aminoethyl-modified MCM41 (NH2-MCM41) according
to the reported procedure,17 namely, the CTAB template method
using aminoethyl triethoxysilane and tetraethoxysilane (see
Supporting Information). We then confirmed that it can encap-
sulate a phosphorylated serin appended coumarin (P-coum 2)
in acidic aqueous solution (pH 5.0, 28 ( 0.3 nmol mg-1 of
NH2-MCM41). It also became clear that the encapsulated
P-coum 2 was released up to 88% by pH shift from 5 to 10
(Figure 1a). A detailed pH dependence study showed that the
release (%) was sharply enhanced above neutral pH, and the
apparent pKa value was determined as 7.5, suggesting the crucial
role of electrostatic interaction between NH2-MCM41 and
P-coum 2 for the entrapment (Figure 1b). We also confirmed
that the release of P-coum 2 from NH2-MCM41 was triggered
by an anion-exchange reaction. For instance, addition of sucrose
octasulfate (Suc-8S) to the suspension of NH2-MCM41 in
aqueous buffer solution facilitated the release (63%) of P-coum
2 by increasing the Suc-8S content without changing the pH
(pH 5.0) (Figure 2a). It is interesting that, as shown in Figure
2b, the release was efficiently induced by other polysulfates and
polyphosphates such as heparin, chondroitin sulfate, ATP, and
IP6, but not by polycarboxylate (hyaluronic acid), monoanions
(AMP, sialyl lactose, and inorganic phosphate), neutral (mal-
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A R T I C L E S
droplet20 as green fluorescent spots (ca. 1-5 µm in diameter)
due to the bound P-coum 2. It was also shown that
NH2-MCM41 was segregated from the supramolecular fibers
stained by the hydrophobic BODIPY dye 4, indicating that these
two materials can provide microdomains orthogonal to each
other in the semi-wet matrix (Figure 3). When the pH of the
gel matrix was changed from 5 to 10, the green spots became
smeared, suggesting pH-triggered release of P-coum 2 from the
interior of NH2-MCM41 to the aqueous gel space. Similarly,
the intensity of the green fluorescence decreased on addition of
Suc-8S. In this case, we quantitatively evaluated the relative
release ratio using red fluorescent microbeads (10 µm in
diameter) as an internal standard. Figure 4 shows the CLSM
images and the corresponding line profiles of fluorescence
intensity traversing the NH2-MCM41 and the fluorescent
microbeads, before and after Suc-8S addition. Apparently, the
green fluorescence due to P-coum 2 entrapped in the interior
of NH2-MCM41 decreased in its intensity by addition of Suc-
8S and the intensity change (1 - F/F0 ) 0.60 ( 0.06) was
estimated by comparison with the red fluorescent microbeads.
Figure 5 summarizes the fluorescence change caused by various
substances. As in the case of aqueous suspension (Figure 2),
heparin, ATP, and IP6 were effective for releasing P-coum 2
from NH2-MCM41, as well as Suc-8S. By contrast, AMP,
inorganic phosphate, maltoheptaose, and ds-DNA were not
effective. Thus, it is clear that the selective anion-exchange
property of the NH2-MCM41/P-coum 2 composite was retained
even when it was embedded in the supramolecular hydrogel
matrix 1.
Polyanion Triggered and Enzyme-Coupled Translocation
of Fluorescent Probe in NH2-MCM41 Supramolecular
Hydrogel Hybrid. By CLSM observation, we confirmed the
release of P-coum 2 from NH2-MCM41 in the gel matrix.
Unfortunately, we also found that the P-coum 2 did not
accumulate in the gel fibers, but dispersed in the aqueous space
of the gel matrix, resulting in smeared CLSM images. To
facilitate accumulation of the fluorescent probe to the fiber
domains, we next introduced coupled enzymatic hydrolysis of
the anionic phosphate group of the P-coum probe. We already
reported that enzymes can be immobilized in saccharide-based
supramolecular hydrogels without loss of natural activity16 and
that sufficient mobility of small molecules is retained in the
gel matrix.15,16 For efficient accumulation of the fluorescent
probe in the hydrophobic fiber domains after enzymatic hy-
drolysis, we introduced a hydrophobic cyclohexyl group into
the C-terminus of P-coum 2, which gave a new probe P-coum
3.21
Figure 1. (a) Release (%) of P-coum 2 in 200 mM acetate buffer (pH 5.0,
600 µL) or 50 mM CHES buffer (pH 10, 600 µL) from NH2-MCM. (b)
pH-Dependent release profiles of P-coum 2 from NH2-MCM. Conditions:
[P-coum 2@NH2-MCM] ) 0.83 mg mL-1 (0.50 mg), RT.
toheptaose), and cationic substrates (glucosamine). These results
may be ascribed to the polyanionic molecules with phosphate
or sulfate groups being more strongly bound to the cationic
interior of NH2-MCM41 than the less anionic P-coum 2, so
that 2 was replaced by the polyanions. The pore size of
NH2-MCM41, estimated as 2.7 nm from BET measurement
(Supporting Information), offered additional selectivity toward
anionic species. For instance, bulky polyanions such as oval-
bumin and double-stranded DNAs (plasmid DNA and λ-DNA)
were not able to replace P-coum 2, suggesting that the size of
the polyanions is another controlling factor for the anion-
exchange in NH2-MCM41.
Catch and Release Function of Amine-Modified MCM41
in Supramolecular Hydrogel. On the basis of the results of the
polyanion-triggered fluorophore release from NH2-MCM41, we
designed a novel MCM-based fluorescence sensing device by
hybridization with a unique microenvironment of supramolecular
hydrogel.18,19 Scheme 1 illustrates our strategy for the construc-
tion of the MCM-enzyme-supramolecular hydrogel hybrid
sensor. We expected that anion exchange within NH2-MCM41
took place even in the supramolecular hydrogel matrix, owing
to its semi-wet environment similar to aqueous solution. By
accumulation of the fluorescent probe released from
NH2-MCM41 on the hydrophobic fibers of supramolecular
hydrogel, we can induce a fluorescence change within the gel
matrix. Efficient translocation of the probe from the aqueous
gel phase to the fiber may be facilitated by its chemical
transformation catalyzed by the embedded enzymes. The altered
fluorescence is a suitable readout signal that is sensitive to the
character and the amount of added anions. Since we showed
that NH2-MCM41 responded selectively to polyanions in
aqueous solution, a composite of NH2-MCM41 and enzyme
with supramolecular hydrogel may be a unique polyanion-
sensing hybrid material.
We investigated the polyanion-triggered and enzyme-coupled
translocation of P-coum 3 by CLSM observations. Figure 6
shows typical CLSM images of the hybrid gel before and after
treatment with Suc-8S and/or acid phosphatase (ACP). While
the addition of only Suc-8S simply decreased the fluorescence
intensity from the spots, the green fluorescent fibers were clearly
visualized by subsequent addition of ACP. Similar CLSM image
changes occurred by pH shift from 5 to 10 followed by alkaline
phosphatase (AP) treatment (data not shown). We also confirmed
that the translocation of probe 3 never occurred without Suc-
We initially evaluated the catch and release function of
NH2-MCM41 embedded in a supramolecular hydrogel 1
matrix. Using confocal laser scanning microscopy (CLSM), we
clearly observed aggregated NH2-MCM41 in a hydrogel
(18) For reviews on supramolecular hydrogels, see: (a) Estroff, L. A.;
Hamilton, A. D. Chem. ReV. 2004, 104, 1201–1217. (b) de Loos, M.;
Feringa, B. L.; van Esch, J. H. Eur. J. Org. Chem. 2005, 3615–3631.
(c) Molecular Gels: Materials with Self-Assembled Fibrillar Networks;
Weiss, R. G., Terech, P., Eds.; Springer: Dordrecht, The Netherlands,
2006; Chapters 17 and 18. (d) Yang, Z.; Xu, B. J. Mater. Chem. 2007,
17, 2385–2393, and references therein.
(20) Matsumoto, S.; Yamaguchi, S.; Wada, A.; Matsui, T.; Ikeda, M.;
Hamachi, I. Chem. Commun. 2008, 1545–1547.
(21) Similar to P-coum 2, we confirmed that P-coum 3 was encapsulated
into NH2-MCM41 in acidic aqueous solution (pH 5.0, 17 ( 0.9 nmol
mg-1 of NH2-MCM41) and released at basic pH (pH 10, 70 ( 3%);
see Experimental Section for details.
(19) Kiyonaka, S.; Sugiyasu, K.; Shinkai, S.; Hamachi, I. J. Am. Chem.
Soc. 2002, 124, 10954–10955.
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Wada et al.
Figure 2. (a) Dependence of the release (%) of P-coum 2 from NH2-MCM on the concentration of Suc-8S ([Suc-8S] ) 0-1.0 mM (0-600 nmol)) in 200
mM acetate buffer (pH 5.0, 600 µL). (b) Release (%) of P-coum 2 from NH2-MCM upon the addition of various anionic species in 200 mM acetate buffer
(pH 5.0, 600 µL). Conditions: [anion] ) 0.50 mM (300 nmol), [ovalbumin], [dsDNA (λ-DNA)] ) 0.30 mM (180 nmol), [plasmid DNA] ) 0.25 mM (152
nmol), [P-coum 2@NH2-MCM] ) 0.83 mg mL-1 (0.50 mg), RT.
Scheme 1. (a) Chemical Structures of Compounds 1-4, Suc-8S, IP6, Heparin, and Chondroitin Sulfate and (b) Construction and Mechanism
Operating in Fluorescent Dye Encapsulated MCM-Enzyme-Supramolecular Hydrogel Hybrid Sensory System
8S addition or basic pH shift, even in the presence of the
phosphatase enzymes, suggesting that P-coum 3 entrapped in
the interior of NH2-MCM41 is completely protected from
enzymatic hydrolysis. These results imply that the P-coum 3
released by Suc-8S or pH shift was hydrolyzed by the phos-
phatase (ACP or AP) in the aqueous space of the gel matrix,
and subsequently the resulting neutral coumarin, which was
more hydrophobic than anionic P-coum 3, migrated to the
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Figure 3. CLSM images of the hydrogel 1 droplet containing BODIPY 4 and P-coum 2 encapsulated NH2-MCM. The left image shows the localization
of BODIPY 4 (λex ) 488 nm/λem ) 515-570 nm), the middle image shows the localization of P-coum 2 (λex ) 458 nm/λem ) 470-510 nm), and the merge
image shown in the right panel was obtained by summing the left and middle images. Conditions: [1] ) 0.090 wt % (50 mM Tris-HCl buffer, pH 5.0), [4]
) 5.0 µM, [P-coum 2@NH2-MCM] ) 9.0 mg mL-1 (4.5 µg), RT.
visualized using FRET-type emission change, which provides
a hydrogel-based fluorescent sensor selective to polyanions. For
FRET sensing, we embedded a suitable FRET acceptor in the
hydrophobic domain of the supramolecular fibers. Controlled
juxtaposition of the FRET acceptor, a BODIPY derivative 4,
was easily accomplished because of the high orthogonality of
the three microdomains, that is, the cationic interior of MCM41,
the aqueous space of the hydrogel, and the hydrophobic fibrous
domain.
Figure 7 shows CLSM images of the hybrid hydrogels
containing BODIPY 4, AP, and NH2-MCM41 entrapping
P-coum 3. At acidic pH, the hydrophobic BODIPY stained the
supramolecular fibers, whereas the P-coum 3 spots were
separately located within NH2-MCM41. Thus, FRET emission
between coumarin and BODIPY was not observed. After pH
shift from 5 to 10, the segregated spots due to P-coum 3 almost
disappeared and the coumarin fluorescence was localized along
the fibers and overlapped perfectly with the emission from
BODIPY (see the merged image). Importantly, FRET emission
(CLSM images under condition 3) was also obtained along the
fiber domains. Thus, it is clear that the translocation of the
coumarin fluorophore from the interior of NH2-MCM41 to the
supramolecular fiber via the aqueous gel space greatly facilitated
the change in FRET emission.
Figure 4. CLSM images of the hydrogel 1 droplet containing P-coum 2
encapsulated NH2-MCM and 10-µm microbeads (a) before and (b) after
120 min of the Suc-8S addition. Fluorescence intensity profiles along lines
2 and 3 in each image are shown below the images. Conditions: [1] )
0.090 wt % (200 mM acetate buffer, pH 5.0), [Suc-8S] ) 0.42 mM (0.50
nmol), [P-coum 2@NH2-MCM] ) 0.83 mg mL-1 (0.80 µg), RT.
This FRET behavior allowed us to monitor the pH-induced
fluorescence spectral change of the gel spots, as shown in Figure
8a. The emission at 483 nm from coumarin decreased, and the
emission intensity at 513 nm from BODIPY concurrently
increased. A seesaw type of emission change also occurred by
addition of Suc-8S, as shown in Figure 8b. The change was
saturated with increase in the Suc-8S content (inset of Figure
8b), clearly demonstrating the validity of the hybrid hydrogel
as a fluorescent anion-sensing material. Figure 8c summarizes
titration curves of the hybrid gel with various substances. IP6,
Suc-8S, and heparin were detected at a 5 nmol level (1 µL of
5 mM analyte solution), whereas sialyl lactose and glucosamine
were not sensed because of their poor anion-exchange activity.
Also, we confirmed that Suc-8S could be detected even in the
coexistence of other anions such as sialyl lactose, AMP, and
so forth (Figure S4 in the Supporting Information). Interestingly,
we noticed that IP3 and ATP were scarcely detected using this
hybrid hydrogel despite the high anion-exchange activity. This
can probably be ascribed to ACP immobilized in the hybrid
gel rapidly hydrolyzing the phosphoester bonds of IP3 and ATP
so as to reduce their concentrations before anion exchange,
indicative of a masking effect of ACP for such polyphosphates.
As a result, the present NH2-MCM41-ACP hydrogel hybrid
Figure 5. Changes in fluorescence intensity ratio of the hydrogel 1 droplet
containing P-coum 2 encapsulated NH2-MCM upon the addition of anionic
species. Conditions: [1] ) 0.090 wt % (200 mM acetate buffer, pH 5.0),
[anion] ) 1.0 mM (0.50 nmol), [P-coum 2@NH2-MCM] ) 0.83 mg mL-1
(0.80 µg), RT.
hydrophobic gel fibers. It should be noted that all of these events
took place within a single hydrogel droplet.
Polyanion-Selective
Fluorescence
Sensing
by
MCM41-ACP-Hydrogel Hybrid. Such translocation of the
fluorescent probe within the hydrogel matrix can be readily
9
J. AM. CHEM. SOC. VOL. 131, NO. 14, 2009 5325

A R T I C L E S
Wada et al.
Figure 6. CLSM images (λex ) 458 nm) of hydrogel 1 containing P-coum 3 encapsulated NH2-MCM (a) before, (b) after 60 min of Suc-8S addition
([Suc-8S] ) 1.15 mM, 15 nmol), and (c) after 90 min of subsequent ACP addition. Conditions: [1] ) 0.090 wt % (200 mM acetate buffer, pH 5.0), [P-coum
3@NH2-MCM] ) 5.0 mg mL-1 (50 µg), RT.
Figure 7. Low-magnification CLSM (×10 objective) images of hydrogel droplet 1 (left side) containing BODIPY 4 and P-coum 3 encapsulated NH2-MCM
in hexadecane (a) before and (b) after 60 min of adjusting to pH 10 and AP addition, which was conducted by the fusion of another hydrogel droplet 1 (right
side) (left: λex ) 488 nm/λem ) 515-570 nm; right: DIC). (c) CLSM (×100 objective) images of the hydrogel 1 droplet. The series of images in (A) and
(B) corresponds to (a) and (b), respectively. The images of condition 1 show the localization of BODIPY 4 (λex ) 488 nm/λem ) 515-570 nm), and the
images of condition 2 show the localization of P-coum 3 (λex ) 458 nm/λem ) 470-510 nm). The images of condition 3 mainly correspond to FRET
emission from P-coum 3 to BODIPY 4 (λex ) 458 nm/λem ) 515-570 nm), although these partially contain contribution of the direct emission from P-coum
3, and the merged images shown in the right panels were obtained by summing the images of conditions 1 and 2. Conditions: [1] ) 0.090 wt % (50 mM
Tris-HCl buffer, pH 5.0), [4] ) 2.0 µM, [P-coum 3@NH2-MCM] ) 9.0 mg mL-1 (4.5 µg), RT.
showed improved selectivity for polysulfates relative to
polyphosphates.
but that color change was not induced by polysulfates. The color
change from red to blue is due to the cancelation of FRET from
the blue fluorescent coumarin dye to the red fluorescent styryl
dye, which indicates that receptor 12 is released from the
hydrophobic fiber 1 containing 13 upon binding to the
polyphosphates.15a Simple comparison between these two chips
by the naked eye thereby allows us to discriminate polysulfates
such as heparin and Suc-8S from polyphosphates such as IP6
and ATP.
We finally prepared a hybrid material array for rapid and high-
throughput sensing for a variety of substrates. As shown in
Figure 9a, bright emission was observed at spots containing IP6,
heparin, Suc-8S, and chondroitin sulfate, but less bright emission
came from ATP, sialyl lactose, maltopentaose, and others. Naked
eye detection of polyanions was very similar to the titration
curves in its selectivity, demonstrating the utility of the present
hybrid materials for high-throughput polyanion sensing. Figure
9b shows a photograph of the phosphate anion-sensing chip of
1 containing coumarin-appended phosphate receptor 12 and the
amphiphilic FRET acceptor (styryl dye) 13, which was previ-
ously developed by us.15a Apparently, the spot colors of
polyphosphates such as IP6, IP3, and ATP changed to bluish,
Conclusions
We demonstrated that rational combination of the anion-
exchange ability of NH2-MCM41 and the semi-wet supramo-
lecular hydrogel matrix successfully produced unique sensing
materials selective to polyanions such as polysulfate oligosac-
9
5326 J. AM. CHEM. SOC. VOL. 131, NO. 14, 2009

MCM-Enzyme-Supramolecular Hydrogel Hybrid Sensor
A R T I C L E S
Elmer Spectrum One on ATR mode. BET nitrogen sorption
experiments were carried out using a Belsorp Belsorp-18 volumetric
adsorption equipment. Elemental analysis was carried out by the
services at Kyoto University.
Synthesis. The gelator 1 was prepared according to the method
reported previously by us.22 P-coum 2 and 3 were prepared as
shown in Schemes 2 and 3, respectively. The synthetic procedure
for P-coum 2 is described in the Supporting Information. BODIPY
4 was prepared from BODIPY-carboxylic acid23 and GalNAc-
NH2,19 as shown in Scheme 4.
Fmoc-O-benzyloxy-phosphono-L-serin Cyclohexyl Amide
(6). A solution of Fmoc-O-benzyloxy-phosphono-L-serin (300 mg,
0.60 mmol) and benzotriazol-1-yloxy-tris(dimethylamino)phosphate
hexafluorophosphate (BOP, 320 mg, 0.72 mmol) in dry DMF (5
mL) was stirred at 0 °C under Ar atmosphere for 30 min.
Cyclohexylamine (87 µL, 0.72 mmol) was then added to the
solution. The mixture solution was stirred for 2 h, and then the
solvent was removed under reduced pressure. The residue was
dissolved in ethyl acetate (100 mL), and the solution was washed
with aqueous sodium bicarbonate (100 mL), 5% aqueous citric acid
(100 mL), and brine (100 mL). The organic layer was collected
and dried over anhydrous Na2SO4 and filtered. The filtrate was
concentrated to dryness, and the residue was purified by column
chromatography (SiO2, chloroform/methanol ) 4:1), affording
compound 6 as a white solid (136 mg, 39%). 1H NMR (400 MHz,
CDCl3, room temperature): δ 1.61-1.81 (m, 10H), 3.64-3.65 (m,
1H), 4.00-4.09 (m, 2H), 4.18-4.21 (m, 1H), 4.31-4.64 (m, 1H
+ 2H), 4.87 (d, J ) 6.0 Hz, 1H), 7.23-7.80 ppm (m, 5H + 10H);
MALDI-MS obsd 579.93 [M + H]+, calcd 579.22.
O-Benzyloxy-phosphono-L-serin Cyclohexyl Amide (7). A
solution of Fmoc-O-benzyloxy-phosphono-L-serin cyclohexyl amide
6 (136 mg, 0.24 mmol) in 20% piperidine DMF (8 mL) was stirred
at room temperature. After 45 min, the solvent was removed under
reduced pressure. The residue was subjected to precipitation by
diisopropyl ether to afford compound 7 as a white solid (82 mg,
97%). 1H NMR (400 MHz, CD3OD, room temperature): δ
1.11-1.34 (m, 10H), 3.30-3.38 (m, 1H (overlapped with metha-
nol)), 3.52-3.72 (m, 1H), 3.98-4.02 (m, 1H), 4.88-4.91 (d, 2H
(overlapped with H2O)), 7.26-7.38 ppm (m, 5H); MALDI-MS obsd
357.78 [M + H]+, calcd 357.15.
Coumarin-O-benzyloxy-phosphono-L-serin Cyclohexyl (8). A
solution of 7-diethylamino coumarin-3-carboxyric acid (35 mg, 0.16
mmol) and BOP (85 mg, 0.19 mmol) in dry DMF (3 mL) was
stirred at 0 °C under Ar atmosphere for 30 min. O-Benzyloxy-
phosphono-L-serin cyclohexy amide 7 (56 mg, 0.16 mmol) in dry
DMF (5.5 mL) and DIPEA (55 µL, 0.32 mmol) was added to the
solution. The mixture solution was stirred at room temperature for
4 h, and then the solvent was removed under reduced pressure.
The residue was dissolved in ethyl acetate (50 mL), and the solution
was washed with aqueous sodium bicarbonate (50 mL), 5% aqueous
citric acid (50 mL), and brine (50 mL). The organic layer was
collected and dried over anhydrous Na2SO4 and filtered. The filtrate
was concentrated to dryness, and the residue was purified by column
chromatography (SiO2, chloroform/methanol ) 4:1), affording
compound 8 as a yellow solid (38 mg, 40%). 1H NMR (400 MHz,
CDCl3/CD3OD ) 6:1, room temperature): δ 1.13-1.88 (m, 10H),
1.23 (t, J ) 7.2 Hz, 6H), 3.44 (q, J ) 7.2 Hz, 4H), 3.65-3.74 (m,
1H), 4.19-4.20 (m, 2H), 4.86-4.87 (m, 1H), 4.97 (d, J ) 4.8 Hz,
2H), 6.40 (s, 1H), 6.43 (d, J ) 8.8 Hz, 1H), 7.23-7.51 (m, 5H +
1H), 7.50 (d, J ) 9.2 Hz, 1H), 8.76 (s, 1H), 9.90 ppm (d, J ) 6.4
Hz, 1H); ESI-MS obsd 598.4 [M - H]s, calcd 598.24.
Figure 8. Fluorescence spectral changes (λex ) 429 nm) of hydrogel 1
containing BODIPY 4 and P-coum 3 encapsulated NH2-MCM (a) before
and after adjusting to pH 10 and AP addition and (b) before and after Suc-
8S and ACP addition. The inset in (b) shows the fluorescence titration curve
for Suc-8S ([Suc-8S] ) 0-1.4 mM (0-15 nmol)). Conditions: [1] ) 0.090
wt % (50 mM Tris-HCl buffer, pH 5.0, [4] ) 2.0 µM for (a) and 200 mM
acetate buffer, pH 5.0, [4] ) 3.0 µM, [Suc-8S] ) 1.4 mM (15 nmol) for
(b)), [P-coum 3@NH2-MCM] ) 5.0 mg mL-1 (50 µg), RT. (c) Plots of
the relative fluorescence intensity changes (r/r0 - 1 ) (F513/F483)/(F513
/
F483)0 -1) of hydrogel 1 containing BODIPY 4 and P-coum 3 encapsulated
NH2-MCM upon addition of various species and ACP (λex ) 429 nm).
Conditions: [1] ) 0.090 wt % (200 mM acetate buffer, pH 5.0), [4] ) 3.0
µM, [anion] ) 0-6.5 mM (0-60 nmol), [P-coum 3@NH2-MCM] ) 5.0
mg mL-1 (50 µg), RT.
charides. The orthogonal domain formation given by these two
materials and maintaining sufficient mobility of small molecules
are crucial for this sensing system. Coupling of the enzyme
reaction in the hybrid gel efficiently modulates the sensing
efficiency and selectivity. It is envisioned that further function-
alization of the MCM and/or the gel fibers, and a new entry of
various enzymes, may lead not only to sophisticated sensing
materials, but also to intelligent drug delivery systems or other
biomaterials. We are now working along this line.
Experimental Section
General. Unless stated otherwise, all commercial reagents were
used as received. Alkaline phosphatase (from Escherichia coli,
TOYOBO) and acid phosphatase (from potato, SIGMA) were used
as received. Thin layer chromatography was performed on silica
gel 60F254 (Merck). Column chromatography was performed on
silica gel 60N (Kanto, 40-50 µm). Reverse-phase HPLC was
conducted with a Hitachi Lachrom instrument with YMC-pack
ODS-A columns. 1H NMR spectra were obtained on a Varian
Mercury 400 spectrometer with tetramethylsilane or residual
nondeuterated solvents as the internal references. FAB, MALDI-
TOF, and ESI mass spectra were recorded using a Shimadzu
QP5050A (NBA as a matrix), Perseptive Biosystems Voyager DE-
RP instruments (dithranol as a matrix), and Applied Biosystems
API2000, respectively. The absorption and fluorescence spectra
were measured using a Shimadzu UV2550 and a Perkin-Elmer LS55
spectrometer, respectively. Fluorescence spectra of the hydrogel
spots on a slide glass plate were recorded using an Otsuka
Electronics high-sensitivity Spectro multichannel photodetector,
MCPD-7000. TEM images were obtained by using a JEOL JEM-
2010 microscopy. FTIR spectra were recorded by using a Perkin-
P-coum 3. Pd-C (10 mg) was added to the solution of coumarin-
O-benzyloxy-phosphono-L-serin cyclohexyl 8 in choloroform/
methanol (4 mL/8 mL). The mixture solution was stirred at room
(22) Matsumoto, S.; Yamaguchi, S.; Ueno, S.; Komatsu, H.; Ikeda, M.;
Ishizuka, K.; Iko, Y.; Tabata, K. V.; Aoki, H.; Ito, S.; Noji, H.;
Hamachi, I. Chem.-Eur. J. 2008, 14, 3977–3986.
(23) Li, J.-S.; Wang, H.; Cao, L.-W.; Zhang, H.-S. Talanta 2006, 69, 1190–
1199.
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A R T I C L E S
Wada et al.
Figure 9. Photographs of (a) MCM-enzyme-supramolecular hydrogel hybrid sensory chip comprising hydrogel 1 containing BODIPY 4, P-coum 3
encapsulated NH2-MCM, and ACP and (b) phosphate anion-sensing chip of hydrogel 1 containing fluorescent receptor 12 and the FRET acceptor 13 for
anionic species assay. The spotted positions of anions are shown at the bottom. Conditions: Spotted volume ) 10 µL. (a) [1] ) 0.090 wt % (200 mM acetate
buffer, pH 5.0), [4] ) 3.0 µM, [P-coum 3@NH2-MCM] ) 5.0 mg mL-1 (50 µg), [anion] ) 1.5 mM (15 nmol). (b) [1] ) 0.10 wt % (50 mM HEPES buffer,
pH 7.2, 100 mM NaCl), [12] ) 50 µM, [13] ) 30 µM, [anion] ) 0.50 mM (5.0 nmol), RT. See Supporting Information for the structures of the anions. (c)
Chemical structures of 12 and 13.
Scheme 2. Synthesis of P-coum (2)
BODIPY Pentanoic Acid (10). A solution of BODIPY-OSu 9
(100 mg, 0.20 mmol) and 5-aminopentanoic acid (35 mg, 0.30
mmol) in DMF/methanol (3 mL/8 mL) was stirred at room
temperature under Ar atmosphere. After 1 h, the solvent was
removed under reduced pressure. The residue was dissolved in
chloroform (100 mL), and the solution was washed with 5%
aqueous citric acid (100 mL) and brine (100 mL). The organic layer
was collected and dried over anhydrous Na2SO4 and filtered. The
filtrate was concentrated to dryness, and the residue was purified
by column chromatography (SiO2, chloroform/methanol ) 10:1),
affording compound 10 as a red solid (89 mg, 90%). 1H NMR (400
MHz, CDCl3, room temperature): δ 1.41 (s, 6H), 1.56-1.71 (m,
2H + 2H), 2.42 (t, J ) 6.8 Hz, 2H), 2.55 (s, 6H), 3.40 (q, J ) 6.0
Hz, 2H), 4.55 (s, 2H), 5.98 (s, 2H), 6.72 (br s, 1H), 7.05 (d, J )
8.8 Hz, 2H), 7.23 ppm (d, J ) 8.8 Hz, 2H); ESI-MS obsd 520.4
[M + Na]+, calcd 520.23.
BODIPY Pentanoic Acid-OSu (11). A solution of BODIPY
pentanoic acid 10 (44 mg, 0.088 mmol), N-hydroxysuccinic imide
(15 mg, 0.13 mmol), and WSC-HCl (25 mg, 0.13 mmol) in dry
DMF (5 mL) was stirred at room temperature under Ar atmosphere.
After 5 h, the solvent was removed under reduced pressure. The
residue was dissolved in chloroform (50 mL), and the solution was
washed with brine (50 mL). The organic layer was collected and
dried over anhydrous Na2SO4 and filtered. The filtrate was
concentrated to dryness, and the residue was purified by column
chromatography (SiO2, chloroform/methanol ) 30:1 to 20:1),
affording compound 11 as a red solid (48 mg, 92%). 1H NMR (400
MHz, CDCl3, room temperature): δ 1.41 (s, 6H), 1.69-1.73 (m,
2H), 1.81-1.84 (m, 2H), 2.55 (s, 6H), 2.68 (t, J ) 6.8 Hz, 2H),
2.84 (s, 4H), 3.42 (q, J ) 6.4 Hz, 2H), 4.55 (s, 2H), 5.99 (s, 2H),
7.05 (d, J ) 8.4 Hz, 2H), 7.23 ppm (d, J ) 8.4 Hz, 2H).
temperature under H2 atmosphere. After 2 h, Pd-C was removed
by filteration, and the solvent was removed under reduced pressure
to afford compound 3 as a yellow solid (16 mg, 100%). H NMR
1
(400 MHz, DMSO-d6, room temperature): δ 1.09 (t, J ) 6.4 Hz,
6H), 1.13-1.67 (m, 10H), 2.44-2.45 (q, 4H (overlapped with
DMSO)), 3.33-3.44 (m, 1H (overlapped with H2O)), 3.92-3.99
(m, 2H), 4.59-4.63 (m, 1H), 6.58 (s, 1H), 6.75 (d, J ) 9.6 Hz,
1H), 7.62 (d, J ) 8.8 Hz 1H), 7.99 (d, J ) 6.8 Hz, 1H), 8.61 (s,
1H), 9.09 ppm (d, J ) 6.8 Hz, 1H); FAB-HRMS obsd 510.2007
[M + H]+, calcd 510.2005 (C23H33N3O8P).
BODIPY-OSu (9). A solution of BODIPY-carboxylic acid (150
mg, 0.38 mmol), N-hydroxysuccinic imide (65 mg, 0.57 mmol),
and water-soluble carbodiimide hydrochloride (WSC-HCl, 1-ethyl-
3-(3-dimethyl aminopropyl)carbodiimde hydrochloride, 108 mg,
0.57 mmol) in dry DMF (5 mL) was stirred at room temperature
under Ar atmosphere. After 2.5 h, the solvent was removed under
reduced pressure. The residue was dissolved in chloroform (100
mL), and the solution was washed with brine (100 mL). The organic
layer was collected and dried over anhydrous Na2SO4 and filtered.
The filtrate was concentrated to dryness, and the residue was
purified by column chromatography (SiO2, chloroform/methanol
GalNAc-BODIPY (4). A solution of BODIPY pentanoic
acid-OSu 11 (15 mg, 0.025 mmol) and GalNAc-NH2 (9.0 mg,
0.034 mmol) in dry DMF (2 mL) was stirred at room temperature
under Ar atmosphere. After 2 h, the solvent was removed under
reduced pressure. The residue was purified by column chromatog-
raphy (SiO2, chloroform/methanol ) 10:1 to 7:1), affording
1
) 30:1), affording compound 9 as a red solid (136 mg, 53%). H
NMR (400 MHz, CDCl3, room temperature): δ 1.41 (s, 6H), 2.55
(s, 6H), 2.89 (s, 4H), 5.05 (s, 2H), 5.98 (s, 2H), 7.08 (d, J ) 8.8
Hz, 2H), 7.25 ppm (d, J ) 8.8 Hz, 2H).
1
compound 4 as a red solid (14 mg, 76%). H NMR (400 MHz,
CDCl3/CD3OD ) 7:1, room temperature): δ 1.31 (s, 6H), 1.45-1.52
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MCM-Enzyme-Supramolecular Hydrogel Hybrid Sensor
Scheme 3. Synthesis of P-coum (3)
A R T I C L E S
Scheme 4. Synthesis of BODIPY (4)
(m, 2H), 1.55-1.56 (m, 2H), 1.91 (s, 3H), 2.13 (t, J ) 6.8 Hz,
2H), 2.43 (s, 6H), 3.22-3.26 (m, 2H + 2H (overlapped with
methanol)) 3.36-3.54 (m, 2H + 2H), 3.56-3.60 (m, 1H + 1H),
3.63-3.70 (m, 1H + 1H), 4.25 (d, J ) 8.0 Hz, 1H), 4.43 (s, 2H),
5.89 (s, 2H), 6.98 (d, J ) 8.0 Hz, 2H), 7.12 ppm (d, J ) 8.0 Hz,
2H); FAB-HRMS obsd 743.3529 [M]+, calcd 743.3513
(C36H48N5O9F2B1).
Preparation of P-coum
3
Encapsulated NH2-MCM.
NH2-MCM (1.0 mg) was suspended in P-coum 3 solution (103
µM, pH 5.0, 200 mM acetate, 324 µL), and the resultant susupen-
sion was vortexed overnight. The suspension was centrifugated
(4000 rpm for 8 min), and supernatant was removed. The P-coum
3 encapsulated NH2-MCM was washed with 200 mM acetate (pH
5.0, 500 µL) four times. All supernatants were collected, and
unencapsulated P-coum 3 was determined by UV-vis spectra.
P-coum 3 loaded in 1.0 mg of NH2-MCM was determined to be
17 ( 0.9 nmol (49 ( 1%).
Preparation of Supramolecular Hydrogel Containing
Fluorescent Dye 2 Encapsulated NH2-MCM. A suspension of
gelator 1 (1.0 mg) in 200 mM acetate buffer (pH 5.0, 1.0 mL) or
50 mM Tris-HCl buffer (pH 5.0, 1.0 mL) was heated to form
homogeneous solution. This hot solution was added to a suspension
of P-coum 2 encapsulated NH2-MCM in aqueous buffer. The
Preparation of P-coum
2 Encapsulated NH2-MCM.
NH2-MCM (1.0 mg) was suspended in P-coum 2 solution (316
µM, pH 5.0, 200 mM acetate, 100 µL), and the resultant susupen-
sion was vortexed overnight. The suspension was centrifugated
(4000 rpm for 8 min), and supernatant was removed. The P-coum
2 encapsulated NH2-MCM was washed with 200 mM acetate (pH
5.0, 500 µL) four times. All supernatants were collected, and
unencapsulated P-coum 2 was determined by UV-vis spectra.
P-coum 2 loaded in 1.0 mg of NH2-MCM was determined to be
28 ( 0.3 nmol (81 ( 5%).
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A R T I C L E S
Wada et al.
suspension was pipetted, and the resultant homogeneous suspension
was dropped into hexadecane to form gel droplets (0.5-1.0 µL)
for CLSM observations or spotted on a slide glass plate and
incubated to complete gelation in a sealed box with high humidity
at room temperature for 30 min for MCPD measurements.
Preparation of Supramolecular Hydrogel Containing
Fluorescent Dye 3 Encapsulated NH2-MCM and a FRET
Acceptor (BODIPY 4). A BODIPY 4 solution in MeOH (2 mM,
2 or 3 µL) was added to a suspension of gelator 1 (1.0 mg) in 200
mM acetate buffer (pH 5.0, 1.0 mL) or 50 mM Tris-HCl buffer
(pH 5.0, 1.0 mL), and the mixture was heated to form homogeneous
solution. This hot solution was added to a suspension of P-coum 3
encapsulated NH2-MCM in aqueous buffer. The suspension was
pipetted, and the resultant homogeneous suspension was dropped
into hexadecane to form gel droplets (0.5-1.0 µL) for CLSM
observations or spotted on a slide glass plate and incubated to
complete gelation in a sealed box with high humidity at room
temperature for 30 min for MCPD measurements.
was added to the hydrogel spots, and the slide glass was incubated
in a sealed box with high humidity at room temperature for 90
min. The fluorescence spectra of the hydrogel spots were traced
by MCPD at room tempreture. The photographs of the resulting
hydrogel chip were collected by using a digital camera under UV
irradiation (λex ) 365 nm, using a handy lamp equipped with a
cutoff filter (<300 nm and >400 nm)).
Preparation
of
Phosphate
Anion
Recognition
Supramolecular Gel Chips. A styryl dye 13 (30 µM) appended
hydrogel 1 (0.10 wt %) containing coumarin-appended phosphate
receptor 12 (50 µM) on a slide glass plate was prepared according
to the procedure reported previously.15a Anion species solution (5
mM, 1 µL) was added to the hydrogel spots, and the slide glass
plate was incubated in a sealed box with high humidity at room
temperature for 10 min. The photographs of the resulting hydrogel
chip were collected by using a digital camera equipped with a cutoff
filter (<420 nm) under UV irradiation (λex ) 365 nm, using a handy
lamp).
CLSM Observations. The sample was spotted on a glass-bottom
dish (Matsunami, noncoat, 0.15-0.18-mm glass bottom) and
subjected to CLSM observation using an inverted confocal laser
scanning microscope (Olympus FV1000-ASW) equipped with a
543- and 633-nm Helium Neon, 458- and 488-nm multi-Ar lasers
(Coherent Inc.). A 100× NA ) 1.40 oil objective or a 10× NA )
0.40 air objective was employed to obtain images.
pH and Polyanion Responses of Fluorescent Dye
Encapsulated
NH2-MCM-Enzyme-Supramolecular
Hydrogel Hybrids. Hydrogel 1 (0.09 wt %) containing 2 or 3
encapsulated NH2-MCM (9 µg µL-1) on a slide glass plate was
prepared according to the procedure described above. After the pH
was adjusted from 5 to 10 and an anion species solution was added,
alkaline phosphatase (AP, 1 µL, 6 m units µL-1) and acid
phosphatase (ACP, 1 µL, 6 m units µL-1) were added to the hybrid
hydrogel spots, respectively, and the slide glass plates were
incubated in a sealed box with high humidity at room temperature
for 60 min. The fluorescence spectra of the hybrid hydrogel spots
were traced by MCPD at room temperature. For droplet experi-
ments, a hydrogel droplet (0.09 wt %, 200 mM CHES buffer, pH
10) containing AP (0.55 m unit µL-1) was fused with the hybrid
hydrogel droplet and the process was visualized by CLSM
observations.
Acknowledgment. We thank Prof. S. Kitagawa, Dr. T. Uemura,
and Mr. N. Yanai (Kyoto University) for BET measurements, Dr.
K. Kuwata (Kyoto University) for HRMS measurements, and Prof.
Y. Chujo and Dr. A. Narita (Kyoto University) for TEM observa-
tions. We gratefully acknowledge financial support from the JST,
CREST program, and the global COE program, “Integrated
Materials Science” of the Ministry of Education, Culture, Science,
Sports, and Technology (Japan).
Preparation of Polyanion Recognition Supramolecular Gel
Chips. BODIPY 4 (3 µM) appended hydrogel 1 (0.09 wt %)
containing NH2-MCM (5 µg µL-1) on a slide glass plate was
prepared according to the procedure described above. Anion species
solution (15 mM, 1 µL) was added to the hydrogel spots, and the
slide glass was incubated in a sealed box with high humidity at
Supporting Information Available: Synthetic procedures for
NH2-MCM41 and P-coum 2, Figures S1-S5, and complete
author list for refs 2 and 6. This material is available free of
charge via the Internet at http://pubs.acs.org.
room temperature for 60 min. Then, ACP (2 µL, 24 m units µL-1
)
JA900500J
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