Tb3+-triggered luminescence in a supramolecular gel and its use as a fluorescent chemoprobe for proteins containing alanine

Article abstract of DOI:10.1039/c4cc06074a

A tetracarboxylic acid-appended thiacalix[4]arene-based ligand with Tb³⁺ formed a supramolecular gel which showed novel fluorogenic sensor capability for probing alanine and proteins containing alanine. This journal is

Full text of DOI:10.1039/c4cc06074a

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Tb3+-triggered luminescence in a supramolecular
gel and its use as a fluorescent chemoprobe for
proteins containing alanine†
Cite this: Chem. Commun., 2014,
50, 13107
Received 4th August 2014,
Accepted 2nd September 2014
Sung Ho Jung,‡a Ka Young Kim,‡a Dong Kyun Woo,b Shim Sung Leea and
Jong Hwa Jung*a
DOI: 10.1039/c4cc06074a
www.rsc.org/chemcomm
A tetracarboxylic acid-appended thiacalix[4]arene-based ligand with medical diagnostics,7 and telecommunications.8 To date,
Tb3+ formed a supramolecular gel which showed novel fluorogenic Tb3+-containing hydrogels and organogels have been obtained
sensor capability for probing alanine and proteins containing alanine. by noncovalently incorporating these compounds as complexes
into a gelator matrix, i.e., as independent coordination complexes.
Amino acids are key intermediates of primary metabolism in all Tb3+ can also be introduced into self-assembled structures of
biological processes.1 In general, HPLC, MS and NMR methods, organic molecules where it acts as a bridging unit that enables the
advanced microchips, portable instrumentation, and other rapid formation of branched 2D and 3D coordination polymers and
sensing systems have recently been applied in studies related networks. We recently demonstrated that a tetracarboxylic acid-
to proteomics, metabolism, astrobiology, the food industry, appended thiacalix[4]arene formed a supramolecular gel with a
and other fields.2 Because all amino acids exhibit a variety of network structure, which acted as a chemoprobe for specific gases
important functions, new chemoprobes for detection of specific containing chlorine atoms.9 In this communication, we show that
amino acids would be useful tools in basic science and applied a tetracarboxylic acid-appended thiacalix[4]arene-based gel with
technology. For example, histidine, a polar amino acid, has been Tb3+ acts as a selective fluorogenic chemoprobe for alanine and
investigated by various detection methods that use chemoprobes alanine-containing proteins. Herein, we report the preparation
composed of functionalized nanoparticles and chromogenic of a luminescent supramolecular gel with Tb3+ and its applica-
receptors.3 In contrast, there have been few reported detection tions as a novel fluorogenic chemoprobe for alanine and alanine-
methods for alanine, a typical hydrophobic amino acid, and containing proteins.
for proteins containing alanine. D-alanine has been determined
by using high-performance liquid chromatography and gas
chromatography.4 These methods are very sensitive, reliable
and reproducible, but they require long measuring times, use
specialized techniques for sample preparation and analysis, and
are very expensive to perform. Indeed, a selective detection method
for alanine and proteins containing alanine has never been
reported by using a chemoprobe.
Lanthanides (Tb3+) are of specific interest for such applications,
owing to their unique magnetic and photophysical properties,
The tetracarboxylic acid-appended thiacalix[4]arene-based
which can be modulated through ligand design by taking advan- ligand 1, which has a 1,3-alternating structure, was prepared in
tage of their high coordination requirement/number (ca. 8–12).5 two steps (Scheme S1 in the ESI†). Compound 3 was reacted with
Consequently lanthanides have found applications in sensing,6 3-bromo ethyl bromoacetate in the presence of anhydrous cesium
carbonate in acetone. Then, compound 2 was treated with KOH to
1
yield desired product 1, as confirmed by H, 13C NMR spectro-
scopy, ESI-MS, FT IR and elemental analysis. For example, the
1H NMR spectrum of ligand 1 showed the presence of carboxyl
acidic protons as broad singlets at 11.5 ppm, indicating that
the ligand has C2 symmetry. The formation of ligand 1 was also
confirmed by HR-ESI MS with m/z 953.43, which matched the
calculated isotopic distribution pattern of [1 + H]+.
a Department of Chemistry and Research Institute of Natural Sciences,
Gyeongsang National University, Jinju, South Korea. E-mail: jonghwa@gnu.ac.kr;
Fax: +82-055-758-6027; Tel: +82-055-772-1488
b College of Pharmacy and Research Institute of Pharmaceutical Sciences,
Gyeongsang National University, Jinju 660-701, Korea
† Electronic supplementary information (ESI) available: Experimental details and
spectroscopic analysis. See DOI: 10.1039/c4cc06074a
‡ These authors equally contributed.
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The absorption spectrum of ligand 1 (5.0 wt%) was obtained
in DMF/water (6 : 1 v/v%) at pH = 11 as shown in Fig. S1 (ESI†).
The absorption spectrum of 1 with Tb3+ exhibited a band centered at
approximately 272 nm (log e = 3.609), assigned to the p–p* transition.
Ligand 1 could gelate a DMF/water mixture (6 : 1 v/v%) in the
presence of Tb3+, indicative of the formation of a supramolecular
polymer (Fig. S2A, ESI†). The formation of supramolecular gel 1 was
undertaken at various concentrations of Tb3+ (0.5–5.0 equiv.), in
which the gel was maintained for several months at room
temperature. As shown in Fig. S2B, the green photoluminescence
emission of the gels under UV irradiation gradually decreased
as the concentration of Tb3+ increased. In contrast, sol 1 with
Tb3+ was non-emissive under the same conditions (Fig. S3,
ESI†). The loss of photoluminescence intensity of ligand 1 with
increasing concentration of Tb3+ in the gel state was attributed
to the coordination geometry.
To investigate the quantitative photoluminescence properties of
the supramolecular gel with Tb3+, we observed the photolumines-
cence emission spectra of the gels at different concentrations of
Tb3+ (Fig. 1). The photoluminescence spectrum of the gel in the
presence of a small amount (0.2 equivalents) of Tb3+ showed a
strong emission, which gave rise to a characteristic Tb3+ complex
emission at 490, 546, 586 and 623 nm. In contrast, the intensities of
the photoluminescence emissions of gels formed upon addition of
larger amounts (0.3–1.0 equivalents) of Tb3+ gradually decreased.
These results indicated that the high geometric number of Tb3+
changed into a lower geometric number.10
To gain insight into the morphology and possibly to reveal the
orientation of the supramolecular polymers within gels, xerogel
samples were observed by scanning electron microscopy (SEM) as
well as by energy-dispersive X-ray spectroscopy (EDX). The SEM
image of xerogel 1 prepared with Tb3+ showed a nanorod structure
(Fig. 2A), which was approximately 100–120 nm in length. Further-
more, the xerogel prepared with Tb3+, consisting of carbon, oxygen,
sulfur and terbium, was also analyzed by EDX (Fig. 2B), and
demonstrated that the supramolecular gel has efficiently incorpo-
rated Tb3+. Thus, the main driving force to induce the supra-
molecular gel 1 with Tb3+ was the coordination bond.
Fig. 2 (A) SEM image of xerogel 1 (5 wt%) with Tb3+ (1 equiv.). (B) EDX
spectrum of xerogel 1 with Tb3+
.
thermogram was due to the transition of the supramolecular
gel 1 into the solution phase.
To investigate the selectivity for specific amino acids, the
fluorescence intensity changes of supramolecular gels prepared
with Tb3+ were observed upon addition of various amino acids
(0.3 mM). As shown in Fig. S5 (ESI†), with the exception of Ser,
Phe and His, addition of other amino acids maintained the
stability of the supramolecular gel, indicating that the amino acids
coordinated to the Tb3+ center. In particular, the supramolecular
gel 1 exhibited a strong emission upon UV irradiation with the
addition of alanine (Fig. 3A). The addition of other amino acids
such as Asp, Val, Leu, Gly, Tyr, Met and Cys, in contrast, has no
effect on the fluorescence emission changes of the supramolecular
gels. Thus, the supramolecular gel responds selectively to alanine,
a response which can be attributed to its coordination to Tb3+ by
alanine. We also recorded the fluorescence spectra of supramole-
cular gels prepared with Tb3+ upon addition of amino acids (Fig. 3B).
Only in the presence of alanine did we observe four narrow
emission bands for the supramolecular gel, which were attributed
to a 5D4
-
7F5 (546 nm) transition. The addition of alanine
resulted in a linear enhancement of the fluorescence intensities
of the supramolecular gel, indicating that the alanine molecule
became coordinated to Tb3+. The supramolecular gel also
showed excellent sensitivity with alanine at a very low concen-
tration of 0.3 mM. The main binding force for high selective
We investigated the thermally promoted stability of supra-
molecular gel 1 by measuring the transition temperature (Tsol–gel
)
of gel 1 by differential scanning calorimetry (DSC) (Fig. S4, ESI†).
Supramolecular gel 1 showed a sharp phase transition at 115 1C,
consistent with an endothermic reaction. This endothermic
Fig. 3 (A) Photograph of supramolecular gel 1 (5 wt%) with Tb3+ (1 equiv.)
upon addition of amino acids (3 mM) under a UV lamp. (B) Photoluminescence
spectra of supramolecular gel (5 wt%) with Tb3+ (1 equiv.) upon addition of
amino acids (3 mM).
Fig. 1 (A) Photoluminescence spectra of supramolecular gel 1 (5 wt%)
with various concentration of Tb3+ (0–1.0 equiv.). (B) Plot of photolumi-
nescence intensity against mole ratio of [Tb3+/1].
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alanine is the coordination bond between the 1 + Tb3+ complex The CD spectra of thyroglobulin showed the negative signals in
and the carboxyl group of alanine. In addition, the hydrophobic the absence and the presence of supramolecular gel 1 + Tb3+,
interaction would occur between ligand 1 and alanine.
indicating that the structure of thyroglobulin did not change
The SEM image of the supramolecular gel upon addition of upon addition of supramolecular gel 1 + Tb3+.
alanine showed that the fiber structure connected a sphere and
We investigated the rheological properties of supramolecular
a sphere as a pearl necklace (Fig. S6, ESI†). No significant 1 + Tb3+ with and without alanine. Fig. S11 (ESA†) illustrates that
fluorescence changes of gels were observed upon addition of G0 and G00 were almost constant with the increase of the frequency
other amino acids, including Asp, Val, Leu, Gly, Tyr, Met and from 0.1 to 100 rad sꢀ1. In addition, G0 is ca. 3.5 times larger
Cys. In addition, SEM images of supramolecular gel 1 + Tb3+ than G00 over all ranges (0.1–100 rad sꢀ1), suggesting that the
showed no change upon addition of other amino acids such as supramolecular gel 1 + Tb3+ is moderately tolerant to external
Asp, Val, Leu and Gly (Fig. S7, ESI†).
force. Both the storage modulus and the loss modulus of the
To determine if the specificity in the detection of alanine is supramolecular gel 1 + Tb3+ upon addition of 1.0 equivalents of
compromised by complex mixtures of other amino acids, we alanine were similar to those of the supramolecular gel 1 + Tb3+
observed the fluorescence intensity changes of supramolecular gel in the absence of alanine. Furthermore, G0 and G00 values of
1 upon addition of alanine in binary systems (Fig. S8, ESI†). As supramolecular gel 1 + Tb3+ both without and with alanine
expected, the fluorescence changes of supramolecular gels were were almost constant with the increasing dynamic frequency
observed when a solution of alanine was added to the mixture. The sweep. These results indicate that the rheological properties of
findings suggest that the highly selective nature of the fluorescence supramolecular gel 1 + Tb3+ did not strongly depend on the
change of supramolecular gels is due to the high selectivity existence of alanine.
of the assay for alanine. The fluorescence changes of the supra-
In conclusion, a tetracarboxylic acid-appended thiacalix[4]arene-
molecular gel were also observed upon addition of various proteins based supramolecular gel has been prepared in the presence of
containing alanine such as ferritin, aldolase, thyroglobulin, Tb3+ and its sensing properties for proteins containing alanine
chymotrypsinogen, catalase and ribonuclease (Fig. 4A), in moieties have been investigated. The supramolecular gel in the
which the proteins contained different numbers of alanine presence of 1.0 equivalent of Tb3+ exhibited no fluorescence
moieties. As shown in Fig. 4, the addition of chymotrypsinogen emission when excited with UV light. In contrast, upon addition
induced the largest fluorescence change of the supramolecular of alanine to the supramolecular gel, a strong green emission
gel fluorescence emission, indicating that even though some of occurred. Interestingly, proteins containing alanine moieties
the alanine residues are located inside the protein, due to their also induced a strong emission from the supramolecular gel.
hydrophobic properties, the alanines within the proteins could The tetracarboxylic acid-appended thiacalix[4]arene-based supra-
be coordinated to Tb3+. Furthermore, the fluorescence intensity molecular gel can function as a ‘‘turn-on’’ fluorescent chemo-
changes of the supramolecular gel were dependent on the numbers probe for alanine and proteins containing alanine, which is the
of alanine moieties in the proteins as shown in Fig. 4B. A linear first example of detection of alanine with a chemoprobe. Thus,
response between the fluorescence intensities and the concen- the tetracarboxylic acid-appended thiacalix[4]arene-based supra-
tration of thyroglobulin was observed over the range of 0.22 nM molecular gel would have potential applications in biological
and 1.12 nM and indicated a detection limit of ca. 0.1 nM (Fig. 4C), analysis. Furthermore, this concept would be valuable for the
suggesting quantitative binding to Tb3+. As the control experiment development of chemoprobes for proteins.
for protein, we also observed the fluorescence change of supra-
This work was supported by a grant from the NRF
molecular gel 1 + Tb3+ upon addition of protein (DTG), which is not (2012R1A2A2A01002547 and 2012R1A4A1027750) supported
an alanine moiety. As expected, no significant change was observed by the Ministry of Education, Science and Technology, Korea.
upon addition of DTG (Fig. S9, ESI†).
In addition, this work was partially supported by a grant from
We observed CD spectra of thyroglobulin before and after the Next-Generation BioGreen 21 Program (SSAC, grant#:
addition of supramolecular gel 1 + Tb3+ as shown in Fig. S10 (ESI†). PJ009041022014), Rural Development Administration, Korea.
Fig. 4 (A) Photoluminescence spectra of supramolecular gel 1 (5 wt%) with Tb3+ (1 equiv.) upon addition of various proteins containing alanine moiety;
ferritin, aldolase, thyroglobulin, chymotrypsinogen, catalase and ribonuclease. (B) Plot of photoluminescence intensity of numbers of alanine moiety at
546 nm. (C) Plot of photoluminescence intensity of supramolecular gel with Tb3+ upon addition of thyroglobulin (0.22–1.12 nM) at 546 nm.
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