A tetranaphthoimidazolium receptor as a fluorescent chemosensor for phytate

Article abstract of DOI:10.1039/c4cc02036g

A new tetranaphthoimidazolium receptor was synthesized and reported to be a selective fluorescent chemosensor for phytate, myo-inositol hexakisphosphate (IP₆). In a 100% aqueous solution at pH 7.4, chemosensor 1 showed a selective fluorescence enhancement for IP6 over IP₃, phosphates, pyrophosphates, AMP, ADP and ATP. An excimer emission at 465 nm linearly increases in the range of 300 nM to 1 μM with a detection limit of 2.28 × 10^-7 M. In addition, first live cell imaging of IP ₆ has been demonstrated by using a synthetic receptor. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4cc02036g

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A tetranaphthoimidazolium receptor as a
fluorescent chemosensor for phytate†
Cite this: Chem. Commun., 2014,
50, 5851
Minji Lee,a Jong Hun Moon,b Eun Jin Jun,a Gyoungmi Kim,a Yong-Uk Kwon,a
Jin Yong Lee*b and Juyoung Yoon*a
Received 19th March 2014,
Accepted 10th April 2014
DOI: 10.1039/c4cc02036g
www.rsc.org/chemcomm
A new tetranaphthoimidazolium receptor was synthesized and reported
Accordingly, various detection methods for the determination of
to be a selective fluorescent chemosensor for phytate, myo-inositol IP6 have been reported, such as refractive index HPLC analysis of
hexakisphosphate (IP6). In a 100% aqueous solution at pH 7.4, chemo- phytate itself or of its hydrolysis products (inositol and phosphate),6
sensor 1 showed a selective fluorescence enhancement for IP6 over IP3, flow injection-capillary zone electrophoresis,7 and gas chromato-
phosphates, pyrophosphates, AMP, ADP and ATP. An excimer emission at graphy mass spectrometry.8 However, these methods suffer from a
465 nm linearly increases in the range of 300 nM to 1 lM with a time consuming sample pretreatment process or the use of advanced
detection limit of 2.28 Â 10À7 M. In addition, first live cell imaging of instruments. A fluorescence chemosensing approach certainly has
IP6 has been demonstrated by using a synthetic receptor.
advantages over these methods, in particular, the opportunity for
in vivo imaging.9
10
11–13
Inositol 1,4,5-trisphosphate (IP3) is known to be an important second
There have been some efforts to sense IP3 and IP6
via
messenger in intracellular signal transduction processes, which can fluorescence changes. These methods were based on a ligand
also control the cellular Ca2+ concentration.1 myo-Inositol hexakis- exchange in which metal ions were removed from metal complexes
phosphate (phytate, IP6) is a fully phosphorylated form of inositol by IP6. Notably, the Ahn group reported a fluorogenic chemosensing
(Fig. 1), which is found in blood, urine, and intracellular fluids.2 IP6 ensemble for IP6 using an eosine and Cu2+ complex.12 The Han
has been regarded as an antinutrient due to its ability to chelate group, on the other hand, reported a colorimetric sensing system for
essential trace minerals, such as Fe, Zn, and Ca.3 Recent studies have IP6 using a combination of tris-Zn benzene derivative as the receptor
reported beneficial properties of IP6, such as blood-glucose-lowering unit and 11-mercaptoundecylphosphoric acid functionalized gold
and lipid-lowering effects, antioxidative properties and anticancer nanoparticles as the reporter unit.13 Recently, Kubo and a coworker
activities.4 In addition, a previous study suggested that phytic acid is reported fluorescence sensing of IP6 without using metal ions as the
a cofactor in DNA repair by nonhomologous end-joining.5a Another binding sites, in which an isothiouronium-attached polythiophene
report using yeast mutants also suggested that intracellular phytic shows a selective fluorescence quenching effect with IP6 at pH 5.5.14
acid may be involved in mRNA export from the nucleus to the
Imidazolium-based receptors have been actively studied due
cytosol.5b However, the exact physiological roles of intracellular to the unique ionic hydrogen bonding interactions between
phytic acid are still unclear.5c
imidazolium (C–H)+ groups and anions.15 Specifically, fluores-
cent receptors bearing imidazoliums, naphthoimidazolium or
bisbenzoimidazoliums have been recently utilized as selective
fluorescent chemosensors for various anionic targets.16
As described above, previously reported examples utilized metal
ion-complex systems. In most of these systems, metal ions are
removed by IP6 and thus cannot be easily applied to image IP6 in
the cell.
Fig. 1 Chemical structures of IP3 and IP6 employed in this study.
In the current study, we synthesized a new tetranaphthoimid-
azolium receptor 1, which shows a selective fluorescence enhance-
ment with IP6 in a 100% aqueous system at pH 7.4.
Naphthoimidazoliums maintain the unique properties of
imidazolium, such as ionic hydrogen bonding interactions, and
they are inherently fluorescent, so there is no need to introduce
a Department of Chemistry and Nano Science, Global Top5 Research Program,
Ewha Womans University, Seoul 120-750, Korea. E-mail: jyoon@ewha.ac.kr;
Fax: +82-2-3277-3419; Tel: +82-2-3277-2400
b Department of Chemistry, Sungkyunkwan University, Suwon 440-746, Korea.
E-mail: jinylee@skku.edu
† Electronic supplementary information (ESI) available: Experimental details and
supplementary figures and characterization of compounds. See DOI: 10.1039/c4cc02036g additional fluorophores.
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Scheme 1 Synthesis of tetranaphthoimidazolium receptor 1.
For the synthesis of fluorescent chemosensor 1, 1-methyl-
1H-naphtho[2,3-d]imidazole 2 was first synthesized according
to the reported procedure.17 A mixture of 2 and 1,2,4,5-
tetrakis(bromomethyl)benzene in acetonitrile afforded 1 in an
80% yield (Scheme 1). Compound 1 was fully characterized by
high-resolution FAB mass spectroscopy, 1H NMR, and 13C NMR
spectroscopy (ESI†).
Fig. 4 Synthesis of tetranaphthoimidazolium receptor 1 in DMSO-d6–
D2O (9 : 1, v/v).
because of the acidic nature of this proton. However, a down-
field shift of this proton could be clearly observed upon the
addition of IP6. Benzylic hydrogens also displayed a slight
downfield shift. These changes can be attributed to the possible
hydrogen bonding interactions between these protons and
phosphate groups of IP6. On the other hand, there were upfield
shifts for aromatic protons, which are probably due to the
excimer formations between two naphthoimidazolium groups.
To obtain an insight into the binding modes and fluores-
cence behaviors of 1 with IP3 and IP6, we carried out density
functional theory (DFT) and time-dependent DFT (TDDFT)
calculations with the M06-2x functional using a suite of Gaus-
sian 09 programs.18 The optimized structures of 1, 1 + IP3 and
1 + IP6 are shown in Fig. 5. Compound 1 is well stacked with the
naphthoimidazolium moieties at a distance of about 4.8 Å,
which can lead to intramolecular excimer formation. The
phosphate groups of IP3/IP6 and the (C–H)+ of the imidazolium
moieties as well as hydrogen atoms in the alkyl side-chain of
The selectivity of 1 was tested with sodium salts of FÀ, ClÀ,
CH3CO2À, HPO42À, ClO4À, NO3À, HSO4À, pyrophosphate (PPi),
AMP, ADP, ATP, IP3, and IP6 (1 equiv.) in HEPES buffer (0.02 M,
pH 7.4). A relatively small monomeric emission at 370 nm and
large excimer emission at 465 nm were observed in the fluores-
cence spectrum, as shown in Fig. 2. Among these various anionic
analysts, only IP6 showed a selective fluorescence enhancement at
465 nm. On the other hand, simple anions such as, PPi, AMP, ADP
and ATP did not induce any significant fluorescence change
(Fig. 3). Fig. S6 (ESI†) demonstrates the fluorescence titrations of
1 (5 mM) in HEPES buffer (0.02 M, pH 7.4) upon adding 0–1 equiv.
of IP6. A Job plot showed 1 : 1 stoichiometry between 1 and IP6, as
shown in Fig. 2b. An excimer emission at 465 nm linearly increases
in the range of 300 nM to 1 mM with a detection limit of 2.28 Â
10À7 M (Fig. S7, ESI†).
1
The partial H NMR spectra of 1 with IP6 in DMSO-d6–D2O
(9 : 1, v/v) are presented in Fig. 4. An imidazolium C-2 proton
(Ha) appears as a small signal as a result of exchange with D2O
Fig. 2 (a) Fluorescence spectra of 1 (5 mM) in HEPES buffer (0.02 M, pÀH
7.4) upon addition of sodium salts of FÀ, ClÀ, CH3CO2À, HPO42À, NO3
,
HSO4À, PPi, AMP, ADP, ATP, IP3 and IP6 (1 equiv.). (b) Job’s plot of 1 with IP6
in HEPES buffer (0.02 M, pH 7.4).
Fig. 5 Calculated structures and schematics for binding modes for the
complexes of 1 with IP3 and IP6. Hydrogen atoms were omitted for clarity
and dashed red lines indicate interactions of the phosphate with hydrogen
atoms (o2.5 Å).
Fig. 3 Fluorescence changes of 1 (5 mM) with sodium salts of HPO42À, PPi,
AMP, ADP, ATP, IP3 and IP6 (1 equiv.) in HEPES buffer (0.02 M, pH 7.4).
5852 | Chem. Commun., 2014, 50, 5851--5853
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the imidazolium moieties are involved in interacting with IP3 and pyrophosphates, AMP, ADP, ATP and IP3 did not induce any
IP6. The p-stacking distance between naphthoimidazolium moieties significant fluorescence change. The possible binding modes and
was shortened to 4.1 Å and 3.6 Å for 1 + IP3 and 1 + IP6, respectively. fluorescence changes are also explained by theoretical calculations.
In binding with IP6, the naphthoimidazolium moieties are closer to We further showed the first successful in vivo imaging of IP6 in cells
each other than in binding with IP3. In addition, the interplanar by using a relatively simple naphthoimidazolium-based fluorescent
dihedral angle (C1–N1–N2–C2) of 1 + IP3 is calculated to be 341 due to probe. By using this relatively simple receptor, we could obtain
the space for motions, while it remains almost stacked with the reasonable selectivity for IP6 in 100% aqueous solution at pH 7.4.
dihedral angle of 41 in 1 + IP6 due to the limited space as a result of
This research was financially supported by a grant from the
the strong interactions with six phosphates. This structural feature is National Creative Research Initiative programs of the National
consistent with NMR experimental data, and responsible for the Research Foundation of Korea (NRF) funded by the Korean
stronger fluorescence of 1 in binding with IP6 than IP3.
government (MSIP) (No. 2012R1A3A2048814). The work at the
To investigate the fluorescence properties of the receptor Sungkyunkwan University was supported by the NRF grant
upon addition of IP6, TDDFT calculations were performed. The (2007-0056343) funded by MEST. JYL acknowledges the support
important orbital transitions to the excitation and the corres- from KISTI supercomputing center through the strategic support
ponding orbital shapes are shown in Fig. S8 (ESI†). The major program for the supercomputing application research (No. KSC-
transition of 1 comes from HOMO - LUMO + 3 and HOMO À 1 - 2013-C2-027).
LUMO + 2 transitions. Though these orbitals are localized in four
naphthoimidazolium groups, the on-site transition is likely to be
dominated considering the weak fluorescence observed in the
Notes and references
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another one resulting in excimer emission.
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Fig. 6 Confocal fluorescence images of 1 in HeLa cells. (a) No 1 (b) 30 mM
1 after 30 min. (c) 30 mM 1, 5 mM phytic acid (IP6) after 30 min. (d) 30 mM 1,
50 mM phytic acid (IP6) after 30 min. Lower images: bright field. ex 405/em
BP 420–480 nm, scale bar: 10 mm.
This journal is ©The Royal Society of Chemistry 2014
Chem. Commun., 2014, 50, 5851--5853 | 5853