Supramolecular hydrogels based on bola-amphiphilic glycolipids showing color change in response to glycosidases

Article abstract of DOI:10.1039/c2cc37908b

We developed supramolecular hydrogels exhibiting reversible thermochromism concurrently with gel-to-sol transition from four glycolipids. In addition, these gels showed the similar color change in response to glycosidases, which can be employed to construct a colorimetric sensor array chip for sensing glycosidases with the naked eye.

Full text of DOI:10.1039/c2cc37908b

ChemComm
View Article Online
View Journal
COMMUNICATION
Supramolecular hydrogels based on bola-amphiphilic
glycolipids showing color change in response to
glycosidases†‡
Rika Ochi,a Kazuya Kurotani,a Masato Ikeda,b Shigeki Kiyonakaa and
Itaru Hamachi*ac
Cite this: DOI: 10.1039/c2cc37908b
Received 31st October 2012,
Accepted 13th December 2012
DOI: 10.1039/c2cc37908b
www.rsc.org/chemcomm
We developed supramolecular hydrogels exhibiting reversible thermo- Thus, convenient diagnostic tools capable of sensing glycosi-
chromism concurrently with gel-to-sol transition from four glycolipids. dases in a rapid and label-free manner are desirable. Herein we
In addition, these gels showed the similar color change in response to report that unique bola-amphiphilic glycolipids self-assemble
glycosidases, which can be employed to construct a colorimetric sensor to form supramolecular hydrogels capable of exhibiting yellow-
array chip for sensing glycosidases with the naked eye.
to-orange color change along with gel-to-sol transition induced by
the glycosidases, which can be exploited for colorimetric assay of
Supramolecular hydrogels offer great potential for a wide range the relevant enzymatic activities as a semi-wet sensor array chip.
of bio-applications due to their high biocompatibility with the
On the basis of our previous results,8 we carried out the reaction
rational design of nanostructures.1 A variety of supramolecular between monosaccharides (glucose, galactose, and mannose) bear-
hydrogels showing gel-to-sol transition in response to bio-related ing an aniline group at the 1-position and 2,3-dichloromaleimide
stimuli (for example, enzymatic reactions2 and biological mole- derivatives bearing an N-hydroxysuccinimide (NHS) activated ester
cules3) have been reported to date. By contrast, supramolecular (Schemes S1 and S2, ESI‡) to construct a small library of amphiphilic
hydrogels capable of exhibiting distinguishable color change in glycolipids. Contrary to our expectation, a nucleophilic substitution
response to the bio-related stimuli have been rarely explored.2l,m,3b,4 reaction between the 2,3-dichloromaleimide moiety and the
Such changes in macroscopic property (not only gel-to-sol transition aniline group proceeded faster than the condensation reaction of
but also color change) of supramolecular hydrogels are beneficial for the activated ester with the aniline group. As a result, we obtained a
user-friendly bio-sensing materials without any special equipment. unique asymmetric bola-amphiphilic type of glycolipids having a
In particular, the color change enables us to construct a colorimetric newly constructed p-conjugated N-alkyl-2-anilino-3-chloromaleimide
sensor array chip for sensing biomolecules with the naked eye.5
(AAC) moiety, a chromophore showing absorbance around 400 nm,9
Recently, detecting and sensing of various glyco-related enzymes in the middle of the molecules and a sugar and a carboxylic acid as
(for example, glycosyltransferase, glycosidase, and glycokinase), hydrophilic groups at both ends as shown in Fig. 1A.
which catalyze attachment of oligosaccharide units to proteins
The hydrogelation capability of the obtained glycolipids was
and lipids or modification of the oligosaccharide units, have screened using the reverse displacement test tubes, demon-
attracted much attention, because these glycoconjugates are strating that four bola-amphiphilic glycolipids (bGlc-C11, aGlc-
involved in many biological processes.6 For example, b-glucosidase C11, bGal-C11, aMan-C11) are excellent hydrogelators (critical
(bGlc-ase), a representative glycosidase, is known to be a biomarker gelation concentrations (CGC) are 0.1 wt% or less) while a
of Gaucher’s disease or bacterial contamination in drinking water.7 glycolipid (bGlc-C5) bearing a short C5 spacer is not (Fig. S1,
Table S1, ESI‡). Interestingly, we noticed that warm orange
a Department of Synthetic Chemistry and Biological Chemistry, Graduate School of
Engineering, Kyoto University, Katsura, Kyoto, 615-8510, Japan.
E-mail: ihamachi@sbchem.kyoto-u.ac.jp; Fax: +81-75-383-2759;
Tel: +81-75-383-2754
b Department of Biomolecular Science, Graduate School of Engineering,
United Graduate School of Drug Discovery and Medical Information Sciences,
Gifu University, Gifu, 501-1193, Japan
c Core Research for Evolutional Science and Technology (CREST), Japan Science and
Technology Agency (JST), 5 Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan
† Dedicated to Professor Andrew Hamilton on the occasion of his 60th birthday.
‡ Electronic supplementary information (ESI) available: Methods, figures
(Fig. S1–S9), schemes (Schemes S1 and S2), and a table (Table S1). See DOI:
10.1039/c2cc37908b
solutions prepared by heating aqueous dispersions of all four
glycolipid powders in vials turned into bright yellow hydrogels
after cooling, whereas the color of bGlc-C5 solution remained
orange. The gelation process and the yellow-to-orange color change
were thermally reversible (Tgel: bGlc-C11: 78 1C (0.10 wt%), aGlc-C11:
78 1C (0.10 wt%), bGal-C11: 75 1C (0.08 wt%), aMan-C11: 63 1C
(0.09 wt%)), as shown in Fig. 1B and C typically for bGlc-C11.
UV-vis absorption spectral measurement confirmed that the
hydrogel state of bGlc-C11 at 25 1C has an absorption
maximum at 402 nm while the solution state at 80 1C has that
at 411 nm (Fig. 1B, Fig. S2, ESI‡ for temperature dependent
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun.

View Article Online
Communication
ChemComm
Fig. 2 (A) Photograph of the supramolecular hydrogel of bGlc-C11 after the addition
of bGlc-ase ([bGlc-C11] = 0.1 wt% in 0.2 M HEPES (pH 7.2), 100 mL, [bGlc-ase] =
120 units per mL, 20 mL) at room temperature, (B) the corresponding UV-vis
absorption spectral change (black line: 0 h, green line: 3 h, blue line: 6 h, red line:
differential spectrum between 6 h and 0 h).
Fig. 1 (A) Chemical structures of glycolipids, (B) UV-vis absorption spectral
change of the supramolecular hydrogel of bGlc-C11 upon heating (black line:
25 1C, blue line: 80 1C, red line: differential spectrum), (C) photographs of the
supramolecular hydrogel of bGlc-C11 showing reversible thermal gel-to-sol transition
and thermochromism, and schematic illustration of supramolecular hydrogels
consisting of glycolipids exhibiting reversible thermochromism ([bGlc-C11] =
0.1 wt%, 0.2 M HEPES (pH 7.2)).
(aGlc-C11, bGal-C11, and aMan-C11) exhibited selective yellow-
to-orange color change and gel-to-sol transitions only in
absorption spectral change). The differential spectrum between response to the corresponding glycosidases (aGlc-ase, bGal-ase,
the two spectra clearly shows the increase at 467 nm and the and a-mannosidase (aMan-ase), respectively) (Fig. S5, ESI‡). This
decrease at 398 nm by the thermal gel-to-sol transition. The implies that the selectivity of the glycosidases is retained under
blue shifted absorption band in the hydrogel state compared to the semi-wet conditions of the supramolecular hydrogels, and
the solution state suggests that the AAC moieties of bGlc-C11 that more importantly the supramolecular hydrogels of the
could stack in an H-type aggregation mode. These results bola-amphiphilic glycolipids can be used as colorimetric sensors
suggest that the AAC moiety in the glycolipids can act as a for glycosidases. HPLC/ESI-MS analysis confirmed that the
probe to readout the self-assembled state and/or structure of b-glucosidic bond of bGlc-C11 is indeed hydrolyzed upon the
the glycolipid molecules. The circular dichroism (CD) spectrum addition of bGlc-ase (76% consumption after 6 h, Fig. S6
of the hydrogel state of bGlc-C11 showed an intense induced ESI‡).10 Such removal of the hydrophilic glucose group from
CD signal around 400 nm, whereas the CD signal weakened bGlc-C11 should disturb the delicate balance of bola-amphiphi-
significantly in the sol state (Fig. S3, ESI‡). This further supports that lic property to form one-dimensional nanostructures. As a result,
the AAC moiety of bGlc-C11 stacks in chiral environments to form the stacked structure of the AAC moieties in the nanostructures
self-assembled structures in the hydrogel state. Entangled networks collapsed, leading to a color change from yellow to orange.11
of one-dimensional self-assembled nanostructures in the bGlc-C11
Finally, we prepared supramolecular hydrogel array chips by
hydrogel were observed using transmission electron microscopy minimizing the above-mentioned colorimetric gel-based sensor
(TEM) (Fig. S4, ESI,‡ TEM images of the other hydrogels also showed for glycosidases. Being consistent with our previous findings,5g
entangled networks of nanofibers). Overall, Fig. 1C summarizes a it took only 20 min to distinguish the yellow-to-orange color
proposed scheme that the glycolipids self-assemble to form supra- change representing the bGlc-ase activity by using micro-size gels
molecular hydrogels exhibiting reversible thermochromism.
(gel spots), which was much faster than the 4 h needed when using
Interestingly, color change from yellow to orange was observed bulk gels (Fig. 3A). Fig. 3B shows a photograph of the glycosidase
concurrently with the gel breakup (gel-to-sol transition) upon the assay in which the four different glycosidases were added (1 h after
addition of bGlc-ase that selectively cleaves the b-glucosidic bond of the addition of glycosidases). For each glycosidase addition, the
the bGlc-C11 hydrogel, as shown in Fig. 2A. On the other hand, hydrogel spots consisting of the corresponding glycolipids showed
other glycosidases such as a-glucosidase (aGlc-ase) or b-galacto- a color change from yellow to orange (see spots positioned in a
sidase (bGal-ase) induced neither such color change nor gel-to-sol diagonal line from top left to bottom right). This clearly demon-
transition of the bGlc-C11 gel (Fig. S5A, ESI‡).
The slight red-shift of UV-vis absorption of the bGlc-C11 sensing the glycosidase activity visually and rapidly.
hydrogel by bGlc-ase seemed to be a trend similar to that of the In summary, we have serendipitously developed supramolecular
strated that the supramolecular hydrogel array chip is capable of
thermochromism upon heating (vide supra) (Fig. 2B). Indeed, hydrogels from four distinctive glycolipids, which exhibit unique
the differential spectrum, that is the increase at 468 nm and reversible thermochromism induced with gel-to-sol transition.
the decrease at 398 nm by the glycosidase-responsive gel-to- Interestingly, the supramolecular hydrogel showed the yellow-to-
sol transition, clearly showed the same behavior as thermo- orange color change distinguishable by the naked eye in response
chromism. Likewise, the hydrogels of the other glycolipids to glycosidases, which was successfully employed to construct a
c
Chem. Commun.
This journal is The Royal Society of Chemistry 2012

View Article Online
ChemComm
Communication
R. V. Ulijn, J. Am. Chem. Soc., 2006, 128, 1070–1071;
(h) P. K. Vemula, J. Li and G. John, J. Am. Chem. Soc., 2006, 128,
8932–8938; (i) R. J. Williams, A. M. Smith, R. Collins, N. Hodson,
A. K. Das and R. V. Ulijn, Nat. Nanotechnol., 2009, 4, 19–24;
( j) D. Koda, T. Maruyama, N. Minakuchi, K. Nakashima and
M. Goto, Chem. Commun., 2010, 46, 979–981; (k) M. Ikeda, R. Ochi,
A. Wada and I. Hamachi, Chem. Sci., 2010, 1, 491–498; (l) X. Li, J. Li,
Y. Gao, Y. Kuang, J. Shi and B. Xu, J. Am. Chem. Soc., 2010, 132,
17707–17709; (m) J. Gao, W. Zheng, D. Kong and Z. Yang, Soft Matter,
2011, 7, 10443–10448; (n) M. E. Hahn and N. C. Gianneschi, Chem.
Commun., 2011, 47, 11814–11821; (o) M. J. Webber, C. J. Newcomb,
R. Bittonc and S. I. Stupp, Soft Matter, 2011, 7, 9665–9672.
3 (a) Y. Zhang, H. Gu, Z. Yang and B. Xu, J. Am. Chem. Soc., 2003, 125,
13680–13681; (b) Z. Qiu, H. Yu, J. Li, Y. Wang and Y. Zhang, Chem.
Commun., 2009, 3342–3344; (c) C. J. Bowerman and B. L. Nilsson, J. Am.
Chem. Soc., 2010, 132, 9526–9527; (d) M. Ikeda, T. Tanida, T. Yoshii and
I. Hamachi, Adv. Mater., 2011, 23, 2819–2822; (e) M. Yamanaka,
N. Haraya and S. Yamamichi, Chem.–Asian J., 2011, 6, 1022–1025.
4 For supramolecular hydrogels showing pH-responsive gel-to-sol
transition and color change, see: F. Rodrıguez-Llansola, B. Escude,
J. F. Miravet, D. Hermida-Merino, I. W. Hamley, C. J. Cardinb and
W. Hayes, Chem. Commun., 2010, 46, 7960–7962.
5 For recent reviews on supramolecular hydrogel-based sensors for
biomolecules, see: (a) P. Anzenbacher Jr., Y.-L. Liu and M. E. Kozelkova,
Curr. Opin. Chem. Biol., 2010, 14, 693–704; (b) M. Ikeda, R. Ochi and
I. Hamachi, Lab Chip, 2010, 10, 3325–3334; (c) J. Liu, Soft Matter, 2011, 7,
6757–6767, and references cited therein. For recent examples, see:;
(d) S. Kiyonaka, K. Sada, I. Yoshimura, S. Shinkai, N. Kato and
I. Hamachi, Nat. Mater., 2004, 3, 58–64; (e) I. Yoshimura, Y. Miyahara,
N. Kasagi, H. Yamane, A. Ojida and I. Hamachi, J. Am. Chem. Soc., 2004,
126, 12204–12205; ( f ) S. Yamaguchi, I. Yoshimura, T. Kohira,
S.-i. Tamaru and I. Hamachi, J. Am. Chem. Soc., 2005, 127,
11835–11841; (g) S.-i. Tamaru, S. Kiyonaka and I. Hamachi, Chem.ÀEur.
J., 2005, 11, 7294–7304; (h) Y. Koshi, E. Nakata, H. Yamane and
I. Hamachi, J. Am. Chem. Soc., 2006, 128, 10413–10422; (i) A. Wada,
S.-i. Tamaru, M. Ikeda and I. Hamachi, J. Am. Chem. Soc., 2009, 131,
5321–5330; ( j) M. Ikeda, T. Yoshii, T. Matsui, T. Tanida, H. Komatsu
and I. Hamachi, J. Am. Chem. Soc., 2011, 133, 1670–1673; (k) M. Ikeda,
K. Fukuda, T. Tanida, T. Yoshii and I. Hamachi, Chem. Commun., 2012,
48, 2716–2718; (l) S. Bhowmik and U. Maitra, Chem. Commun., 2012, 48,
4624–4626.
Fig. 3 (A) Time course of the color change of the bGlc-C11 supramolecular hydrogel
(a: bulk gels, b: gel spots) upon addition of bGlc-ase (gel spots: [bGlc-C11] = 0.1 wt% in
0.2 M HEPES (pH 7.2), 10 mL, glycosidases: [bGlc-ase] = 120 units per mL, 2 mL) at room
temperature (see Fig. S9 (ESI‡) for details). Error bars represent standard devia-
tions (n = 3). (B) Colorimetric glycosidase activity assay using a supramolecular
hydrogel-based sensor array chip (gel spots: [bGlc-C11], [bGal-C11] = 0.5 wt%,
[aGlc-C11], [aMan-C11] = 1.0 wt% in 0.2 M HEPES (pH 7.2), 10 mL, glycosidases:
[glycosidase] = 120 units per mL, 2 mL, at room temperature).
colorimetric sensor array chip for rapid detection of glycosidases.
We believe that this sensor platform consisting of glyco-related
enzyme substrates as self-assembling components may be extended
to sensing materials for glycokinase and glycosyltransferase, whose
assay requires tedious and indirect manners.12,13 Further research
along these lines is under progress.
6 A. Varki, Glycobiology, 1993, 3, 97–130.
This work was supported in part by the JST (Japan Science
and Technology Agency), CREST program. We acknowledge
Prof. S. Kitagawa, Prof. T. Uemura, and Mr T. Kaseda (Kyoto
University) for XRD and Dr K. Kuwata (Kyoto University) for MS
measurements.
7 (a) C. M. Davies and S. C. Apte, Environ. Toxicol., 1999, 13, 355–359;
(b) L. B. Daniels and R. H. Glew, Clin. Chem., 1982, 28, 569–577;
(c) M. Manafi, W. Kneifel and S. Bascomb, Microbiol. Rev., 1991, 55,
335–348.
8 M. Ikeda, R. Ochi, Y.-s. Kurita, D. J. Pochan and I. Hamachi,
Chem.–Eur. J., 2012, 18, 13091–13096.
9 (a) L. Hanaineh-Abdelnour, S. Bayyuk and R. Theodorie, Tetrahe-
dron, 1999, 55, 11859–11870; (b) W. H. Watson, G. Wu and
M. G. Richmond, J. Chem. Crystallogr., 2004, 33, 983–988.
Notes and references
10 Careful examination of ESI-MS spectra revealed that dechlorination
of the AAC moiety took place during the gel breakup, which should
also contribute to the color change (see Fig. S7, ESI‡).
1 For recent reviews on supramolecular hydrogel, see: (a) L. A. Estroff
and A. D. Hamilton, Chem. Rev., 2004, 104, 1201–1217; (b) M. de Loos,
B. L. Feringa and J. H. van Esch, Eur. J. Org. Chem., 2005, 3615–3631;
(c) A. R. Hirst, B. Escuder, J. F. Miravet and D. K. Smith, Angew. Chem.,
Int. Ed., 2008, 47, 8002–8018.
2 For recent reviews on enzyme-responsive supramolecular hydrogel,
see: (a) R. J. Mart, R. D. Osborne, M. M. Stevens and R. V. Ulijn,
Soft Matter, 2006, 2, 822–835; (b) Y. Gao, Z. Yang, Y. Kuang,
M.-L. Ma, J. Li, F. Zhao and B. Xu, Pept. Sci., 2010, 94, 19–31. For
recent examples, see; (c) B. H. Hu and P. B. Messersmith, J. Am.
Chem. Soc., 2003, 125, 14298–14299; (d) Z. Yang, H. Gu, D. Fu,
P. Gao, J. K. Lam and B. Xu, Adv. Mater., 2004, 16, 1440–1444;
(e) H.-W. Jun, V. Yuwono, S. E. Paramonov and J. D. Hartgerink, Adv.
Mater., 2005, 17, 2612–2617; ( f ) K. J. C. van Bommel, M. C. Stuart,
B. L. Feringa and J. van Esch, Org. Biomol. Chem., 2005, 3,
2917–2920; (g) S. Toledano, R. J. Williams, V. Jayawarna and
11 TEM observation showed the loss of 10 mm-long nanofibers and their
network after the addition of bGlc-ase (see Fig. S8, ESI,‡ the loss of
long nanofibers and their network in the other hydrogels after the
addition of the corresponding glycosidases was also observed).
12 For examples of glycokinase assay, see: (a) K. Uyeda and E. Packer,
J. Biol. Chem., 1965, 240, 4682–4688; (b) E. A. Newsholme,
J. Robinson and K. Taylor, Biochim. Biophys. Acta, 1967, 132,
338–346; (c) W. D. Scheer, H. P. Lehmann and M. F. Beeler, Anal.
Biochem., 1978, 91, 451–463.
13 For recent reviews on glycosyltransferase assay, see: (a) M. M. Palcic
and K. Sujino, Trends Glycosci. Glycotechnol., 2001, 13, 361–370;
(b) G. K. Wagner and T. Pesnot, ChemBioChem, 2010, 11, 1939–1949;
(c) M. M. Palcic, Curr. Opin. Chem. Biol., 2011, 15, 226–233.
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun.