Responsive two-photon induced europium emission as fluorescent indicator for paralytic shellfish saxitoxin

Article abstract of DOI:10.1021/ol2018246

A water-soluble europium(III) complex (1) has been synthesized and demonstrated to be a specific fluorescence probe for the paralytic shellfish toxin saxitoxin, a neurotoxin that blocks the voltage-gated sodium channels on cell membranes. Saxitoxin binds

Full text of DOI:10.1021/ol2018246

ORGANIC
LETTERS
2011
Vol. 13, No. 19
5036–5039
Responsive Two-Photon Induced
Europium Emission as Fluorescent
Indicator for Paralytic Shellfish Saxitoxin
Shuzhong He,† Hongguang Li,† Yuk-Wang Yip,‡ Chi-Tung Yeung,‡ Yuen On Fung,‡
Hoi-Kuan Kong, Ho-Lun Yeung,‡ Ga-Lai Law,‡ Ka-Leung Wong,§ Chi-Sing Lee,*,†
Michael Hon-Wah Lam,*, Margaret B. Murphy, Paul Kwan-Sing Lam, and
Wing-Tak Wong*,‡,^
Laboratory of Chemical Genomics, School of Chemical Biology and Biotechnology,
Peking University Shenzhen Graduate School, Shenzhen University Town, Xili,
Shenzhen 518055, China, Department of Applied Biology and Chemical Technology,
The Hong Kong Polytechnic University, Hung Hom, Hong Kong SAR, Department of
Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong SAR,
Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue,
Kowloon Tong, Hong Kong SAR, and State Key Laboratory for Chirosciences,
The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong SAR
lizc@szpku.edu.cn; bhmhwlam@cityu.edu.hk; bcwtwong@polyu.edu.hk
Received July 7, 2011
ABSTRACT
A water-soluble europium(III) complex (1) has been synthesized and demonstrated to be a specific fluorescence probe for the paralytic shellfish
toxin saxitoxin, a neurotoxin that blocks the voltage-gated sodium channels on cell membranes. Saxitoxin binds to the europium complex
(KB = 6.1 ꢀ 104 Mꢁ1) and triggers a two-photon induced fꢁf emission enhancement by over 100% and increases the two-photon absorption cross-
section from 9 to 36 GM.
There is an increasing public concern about the contam-
ination of seafood and drinking water resources by algal
toxins. Recent studies in Southeast Asian and Pacific
waters have identified algal toxins as one of the major
threats to the local fisheries, aquaculture industry, marine
environmental quality, and public health of the region.
Chemical detection of algal toxins in natural water is an
analytically challenging task, as these toxins are cationic
and highly hydrophilic and do not possess any chromo-
phores or fluorophores.1
Among the various aquatic biotoxins, paralytic shellfish
poisoning (PSP) toxins are probably the most difficult
to detect in natural water. The neurotoxins responsible
for PSP are saxitoxin (STX) and its analogues, such as
† School of Chemical Biology and Biotechnology, Peking University
Shenzhen Graduate School.
‡ Department of Applied Biology and Chemical Technology, The Hong Kong
Polytechnic University.
Department of Biology and Chemistry, City University of Hong Kong.
§ Department of Chemistry, Hong Kong Baptist University.
^ State Key Laboratory for Chirosciences, The Hong Kong Polytechnic
University.
~
(1) Fonfrıa, E. S.; Vilarino, N.; Campbell, K.; Elliott, C.; Haughey,
´
S. A.; Ben-Gigirey, B.; Vieites, J. M.; Kawatsu, K.; Botana, L. M. Anal.
Chem. 2007, 29, 6303–6311.
r
10.1021/ol2018246
Published on Web 09/06/2011
2011 American Chemical Society

neosaxitoxin and decarbamoylsaxitoxin.2 They belong toa
class of highly potent algal neurotoxins that owe their
toxicity to their ability to block voltage-gated sodium
channels on cell membranes.3 There are several methods
to detect STX, such as high-performance liquid chromato-
graphy (HPLC) coupled with chemical derivatization or
mass spectrometric (MS) detection. The postcolumn fluor-
escent derivatization HPLC method by Oshima et al. is the
most widely adopted analytical protocol for PSP deter-
mination.4 However, the current analytical process is
tedious, and the results obtained are not always reprodu-
cible. Immunoassays are another alternative to the chemi-
cal qualitative determination of PSP toxins. The major
drawback is that it requires toxin-specific antibodies,
which are expensive and have short storage lives. This
renders the immunoassay incapable of screening more
than a few PSP toxin variants.5 Thus, a molecular sensor
that can directly respond to PSP toxins in water would be
useful.
Stokes shifts, and sharp emission peaks.7 In addition, there
are various examples of 4fNꢁ4fN luminescence sensitized
by the antenna effect following multiphoton absorption of
tripodal amide ligands.8 These phenomena are particularly
useful for addressing the low signal-to-noise ratios in
existing STX in situ tracers. Herein, we report the devel-
opment of a novel water-soluble emissive europium com-
plex (1), which can be excited in the biologically relevant
near-infrared region through multiphoton absorption, for
STX sensing.
Scheme 1. Synthesis of Europium Complex 1
Figure 1. Structure of europium complex 1 and saxitoxin.
One of the promising alternatives to the existing detec-
tion methods is chemosensing, which enables real-time and
in situ monitoring of an analyte. The molecular sensing
elements involved are generally robust and reusable. Re-
cently, several optical chemosensors featuring the diaza-
18-crown-6 moiety demonstrated their specificity for
STX.6 However, the emission bands of these materials
for STX are usually broad with short lifetimes in the
nanosecond range and have low signal-to-noise ratios.
Incorporation of lanthanides is an attractive approach
for developing STX sensors since lanthanide complexes
generally exhibit long-lived luminescence lifetimes, large
As shown in Figure 1, europium complex 1 contains an
amide-substituted 1,4,7,10-tetraazacyclododecane ligand
and its pendant arms with carboxylic acid groups, a highly
conjugated organic chromophore and a diaza-18-crown-6
derivative. The carboxylic groups should help to improve
the solubility of the complex for the detection of STX in
water/food samples, and the diaza-18-crown-6 moiety
should provide the binding site for “trapping” the STX.
Complex 1 was readily prepared from the 2-bromoace-
tamide (2).9 As shown in Scheme 1, monoalkylation of
€
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Org. Lett., Vol. 13, No. 19, 2011
5037

diaza-18-crown-6 with 2 using sodium bicarbonate in
acetonitrile at 50 ꢀC afforded 3 in 57% yield. Amide
coupling of 3 with acid 410 finished the synthesis of ligand
absolute quantum yield (Φ) and the emission lifetime (τ)
of complex 1 are 0.03 and 0.56 ms, respectively. The two-
photon absorption cross section of complex 1 is around 9
GM (GM = 10ꢁ50 cm4 s photonꢁ1 moleculeꢁ1).
1
5, which is fully characterized by H and 13C NMR and
HRMS. After acid hydrolysis of the tert-butyl esters with
TFA, the crude product was treated with Eu(III) triflate.
Recrystallization of the crude complex with CH3CN
afforded 1 in 50% yield. The structure of europium com-
plex 1 was characterized unambiguously by HRMS.
When complex 1 was titrated against STX in water, a
significant enhancement of the luminescence signals was
observed with both linear and two-photon excitation
(Figure 2), indicating complex formation between complex
1 and STX. After complex 1 binds with STX, the emission
intensity and quantum yield increased by more than 50%
(Φ = 0.08). The binding ratio, constant and rate between
complex 1 and STX have been determined by the hyper-
sensitive 5D0 f 7F2 emission intensity at various concen-
trations of STX. Although the binding constant of
complex 1 (KB = 6.1 ꢀ 104 Mꢁ1) is smaller than that
reported by Gawley et al., complex 1 demonstrated long
lifetime emission and its potential for developing as a time-
resolved detection system and aim for the improvement of
signal-to-noise ration during the sensing process.6 More-
over, an observable color change from pale yellow to red
was observed upon binding of STX in aqueous solutions
(insert of Figure 3).
Figure 2. Enhancement of europium emission intensities of
complex 1 (50 μM in water) upon binding with STX: (a) linear
excitation, λex = 350 nm, [STX] = 5ꢁ100 μM; (b) two-photon
excitation, λex = 750 nm. Squares: [STX] = 0 (9 GM), circles:
[STX] = 15 μM (36 GM). The inset of (b) shows the power
dependence of two-photon induced fꢁf emission of complex 1
upon boning with STX.
Figure 3. Binding isotherms for titration of STX against euro-
7
pium emission (5D0 f F2) response for complex 1 (50 μM in
water). The curve fit assumes a 1:1 binding stoichiometry. (Kb =
6.1 ꢀ 104 Mꢁ1). The inset shows the observable color change of
complex 1 upon binding with STX in aqueous solutions.
Since UV excitation has the disadvantages of low signal-
to-noise ratio and toxicity in situ detection, two-photon
induced emission is one of the promising solutions to these
problems. Europium complex 1 demonstrated real-time
two-photon induced fꢁf emission response to STX
(Figure 2b). STX serving as the trigger for the two-photon
induced fꢁf emission of complex 1. After “trapping” the
STX by the crown moiety, the electron localization in the
conjugated system is changed probably due to hydrogen
bondings and electrostatic interactions, which led to a
dramatic increase of the two-photon induced emission
intensity (three times) with two-photon absorption cross
section increaed from 9 to 36 GM (Figure 2b).
With complex 1 prepared, its photophysical properties
in water were studied. The conjugated chromophore of 1
demonstrated a strong two-photon absorption and appro-
priate triplet states (21739 cmꢁ1) which provide an efficient
pathway for the energy transfer from the ligand to the
excited state (5D0) of the europium. Four structural red fꢁf
(5D0 f 7FJ, J = 1ꢁ6 Hz) emission bands can be obtained
from complex 1 upon excitation at 350 and 750 nm. The
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Z. M. J. Am. Chem. Soc. 2006, 128, 15072–15073.
5038
Org. Lett., Vol. 13, No. 19, 2011

The luminescence lifetime, quantum yield, and the num-
ber of water molecules in the first coordination sphere of
complex 1 are summarized in Table 1. There are more
water molecules directly bound to the chelated europium
metal in the absence of STX than in the presence of STX.
The number of inner-sphere water molecules decreases
from 0.84 to 0.1 upon addition of STX, and the result is
in good agreement with the change in hypersensitive
europium emission in an aqueous environment.11
that is less phototoxic and can visualize a specific toxin
within the “biological window” of excitation (700ꢁ1000
nm). The utility of the tracer in biological systems will be
explored both in vitro and in vivo. Our results demonstrate
the great potential of the europium complex (1) as a new
generation of biotracer for STX, as they have strong bind-
ing affinity (KB = 6.1 ꢀ 104 Mꢁ1) and long lifetime emis-
sion. This tracer could find applications both for STX detec-
tion for research purposes as well as in the marketplace.
Table 1. Luminescence Lifetimes (τ), Number of Inner-Sphere
Water Molecules (q), Quantum Yield (j), and Two-Photon
Absorption Cross-Section (δ) of Complex 1 (50 μM in Water) in
the Presence and Absence of STX
complex
τ(H2O)
τ(D2O)
qa
j(H2O)b
δ/GM (H2O)c
1
0.56
0.87
1.23
1.22
0.84
0.1
0.03
0.08
9
1 þ STX
36
a Derived hydration numbers, q ((20%) qEu = 1.2[(k(H O)
ꢁ
2
k(D O)) ꢁ (0.25 þ 0.07x)] (x = number of carbonyl-bound amide NH
2
oscillators),12 decay curve monitored at 616 nm (5D0 f7F2, λex
=
350 nm).10 b λem = 560ꢁ700 nm, λex = 350 nm. c Two-photon absorp-
4
tion cross-section (GM = 10ꢁ50 cm s photonꢁ1 moleculeꢁ1, λem
=
580ꢁ700 nm, λex = 750 nm).
The selectivity of europium complex 1 for STX over
other bioactive cations (Kþ, Naþ, Ca2þ) and motif PSPs
(neosaxitoxin, decarbamonylsaxitoxin, and decarbamonyl-
neosaxitoxin) havebeen determined. Asshown in Figure 4,
complex 1 shows high specificity toward STX. These
results suggested that the carbamate and the guanidine
moieties of STX are both important for binding with
complex 1. This selectivity is particularly important be-
cause these cations in food samples can bind competitively
with selectivity crown ethers and interfere with the re-
sponse of cations. The fꢁf emission enhancement of com-
plex 1 for STX is greater than that for other common
bioactive cations. The significant fꢁf emission enhance-
ment can be visualized under UV-excitation after complex
1 binds with STX (Figure 2).
Figure 4. Response of europium emission variation (5D0 f 7F2)
of complex 1 (10ꢁ5 M) to different PSP toxins (15 μM) and metal
ions 60 μM, λex = 350 nm).
Acknowledgment. This work was funded by grants
from The Hong Kong Research Grants Council, the Hong
Kong Polytechnic University (PolyU 503211P), City Uni-
versity of Hong Kong (CityU3/CRF/08), Peking Univer-
sity Shenzhen Graduate School, and Hong Kong Baptist
University (FRG 1/10-11/037).
In conclusion, we have designed and synthesized euro-
pium complex 1 with a modified STX diaza-18-crown
binding site which was demonstrated to be highly specific
and responsive europium emission for STX through linear
and nonlinear (near-infrared) excitation. The binding of
STX triggered and enhanced the two-photon-induced
visible fꢁf emission of the europium complex. Based on
these findings, we can further establish a new STX tracer
Supporting Information Available. Experimental de-
tails of synthesis, NMR spectra and HRMS of compound
3 and ligand 5, and HRMS spectra of complex 1. This
material is available free of charge via the Internet at
http://pubs.acs.org.
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Org. Lett., Vol. 13, No. 19, 2011
5039