Development of off-on fluorescent probes for heavy and transition metal ions

Article abstract of DOI:10.1039/b924503k

A carbonyl group was positioned between 1,8-naphthalimide and di-2-picolylamine (DPA) and played a key role of displaying fluorescence enhancements with heavy and transition metal (HTM) ions through increasing the oxidation potential of the fluorophore, blocking HTM ions from sterically interacting with the naphthalimide fluorophore, and by acting as a sacrificial donor.

Full text of DOI:10.1039/b924503k

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Development of off–on fluorescent probes for heavy and
transition metal ionsw
Zhaochao Xu,abc Su Jung Han,b Chongmok Lee,b Juyoung Yoon*bd and David R. Spring*a
Received (in Cambridge, UK) 23rd November 2009, Accepted 14th December 2009
First published as an Advance Article on the web 12th January 2010
DOI: 10.1039/b924503k
A carbonyl group was positioned between 1,8-naphthalimide and
di-2-picolylamine (DPA) and played a key role of displaying
fluorescence enhancements with heavy and transition metal
(HTM) ions through increasing the oxidation potential of the
fluorophore, blocking HTM ions from sterically interacting
with the naphthalimide fluorophore, and by acting as a
sacrificial donor.
the close proximity of the HTM ions to the fluorophore. For
example, Ghosh’s cryptand receptor blocks HTM ions from
sterically and electronically interacting with the peripheral
fluorophore.6a In a similar manner, the twisted and thus
largely decoupled benzene ring in Rurack’s system6b and the
twisted piperazine ring in Xiao’s probe6c lead to high FE. The
second approach is to increase the oxidation potential of the
fluorophore. This should reduce the rate of single electron
transfer (SET) from its singlet excited state to HTM ions and,
thereby, make quenching less efficient.7 The probe developed
by Ramachandram and Samanta relies on this strategy.7
The third approach is to introduce sacrificial donors that
participate in SET with the HTM ions instead of the
fluorophore. In previous studies, it was found that two
unprotonated aromatic nitrogens act as electron donors to
Cu2+ to avoid quenching the fluorescence.8
Heavy and transition metal (HTM) ions play an important
role in many biological and environmental processes.1 For
example, Zn2+ and Cu2+ play essential roles in biochemical
reactions like catalysis, transport or biosynthesis at trace level
(o1 mM). Some heavy metal ions, like Cd2+ or Hg2+, are
potentially carcinogenic or mutagenic and many HTM ions
affect the toxicity of organic xenobiotics through interaction
with metabolizing enzymes or protein synthesis. Fluorescent
probes are powerful tools to monitor in vitro and/or in vivo
HTM ions because of the simplicity and high sensitivity of
fluorescence. The fluorescence enhancement (off–on signal)
induced by complexation with HTM ions is more desirable
than fluorescence quenching (on–off signal) in terms of
increased sensitivity and selectivity. However, the develop-
ment of off–on fluorescent probes for HTM ions remains a
significant challenge.2 A typical fluorescent probe for HTM
ions contains a linked fluorophore (the signal source) to a
receptor (the recognition site).3 Unfortunately, fluorescence
quenching is usually observed when HTM ions are bound to
probes of this type due to quenching by heavy atom enhanced
intersystem crossing (e.g. Hg2+)4 or energy/electron transfer
(e.g. paramagnetic Cu2+).5 Herein we present three general
strategies for surmounting these hurdles. Moreover, the design
and synthesis of a probe based on these strategies is described,
In this report, we designed a new sub-class of probe by
combining 1,8-naphthalimide (fluorophore) with di-2-picolyl-
amine (DPA, receptor), which utilized photoinduced electron
transfer (PET) as the mechanism for signal induction (Fig. 1).
It was found that HTM ions quenched fluorescence of 1 in
acetonitrile (Fig. 2) and aqueous solution (CH3CN–HEPES =
1 : 9, HEPES 0.5 M, pH = 7.4) (Fig. S1, ESIw).9 In order to
block quenching of the 1,8-naphthalimide singlet excited state
by bound HTM ions, inspired by the role it plays in the
spirolactam ring-opening process of rhodamine-based
probes,10 a carbonyl group was positioned between the
naphthalimide and DPA to generate probe 2. The electron-
withdrawing carbonyl increases the oxidation potential of the
fluorophore. In addition, the carbonyl oxygen can coordinate
HTM ions along with DPA and then may block HTM ions
from sterically interacting with the fluorophore and play the
role of a sacrificial donor. Also, the amide linkage can increase
the inflexibility of the receptor.11 The fluorescence spectrum of
2 in acetonitrile contains an emission band with a maximum at
481 nm. The presence of the amide group in 2 decreases the
electron donating ability of the conjugated NH nitrogen and
therefore results in ca. 40 nm blue shift in emission compared
to that of 1 (520 nm).12 Addition of HTM ions to acetonitrile
solutions of 2 causes an increase in fluorescence intensity due
to PET, the extent to which depends on the nature of the ions,
and a shift in the emission maximum from 481 to ca. 430 nm
due to internal charge transfer (ICT) (Table 1 and Fig. S2–S11,
ESIw). The dependence of the ratio of intensities of emission at
430 nm to that at 481 nm (I430/I481) on the nature of the HTM
ions indicates that the stoichiometry of 2–Mn+ complexes is
1 : 1. The binding of HTM ions to the carbonyl oxygen causes
a further decrease in the electron donating ability and a blue
shift in the emission maximum (Fig. S2–S11, ESIw).12 Thus we
which displays
HTM ions.
a fluorescence enhancement (FE) with
In order to prevent fluorescence quenching and preserve the
ability for FE to take place upon binding of the HTM ions, at
least three strategies are available. One approach is to prevent
a Department of Chemistry, University of Cambridge, Lensfield Road,
Cambridge, UK. E-mail: spring@ch.cam.ac.uk
b Department of Chemistry and Nano Science, Ewha Womans
University, 120-750, Seoul, Korea. E-mail: jyoon@ewha.ac.kr;
Fax: +82-2-3277-3419
c State Key Laboratory of Fine Chemicals, Dalian University of
Technology, Dalian 116012, China
d Department of Bioinspired Science, Ewha Womans University,
120-750, Seoul, Korea
w Electronic supplementary information (ESI) available: Synthesis of
compound 1 and 2. Fluorescence and UV-Vis absorption spectra of 1
and 2 with metal ions. CCDC 752644. For ESI and crystallographic
data in CIF or other electronic format see DOI: 10.1039/b924503k
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Table 1 Maximum of fluorescence enhancement (FE) and emission
wavelength changes of 2 in the presence of HTM ions in acetonitrilea
lem/nm
FEb
lem/nm
FE
None
Cr3+
Fe2+
Fe3+
Co2+
Ni2+
481
440
427
430
430
435
1
Cu2+
Zn2+
Ag+
430
430
446
430
435
427
3.8
46
1.6
36
82
31
58
2.7
7.6
2.8
3.1
Cd2+
Hg2+
Pb2+
Fig. 1 Structures of the fluorescent probes studied.
can see that the introduction of the carbonyl group in 2
protects fluorescence from quenching by HTM ions. The blue
shift in emission can provide a ratiometric fluorescence assay
for HTM ions.
a
Experimental conditions: [2] = 10 mM, [Mn+] = 30 mM, lex
=
b
360 nm at 25 1C. Relative quantum yield (FF) in comparison to 2 in
the absence of HTM ions. Calculated by comparison of corrected
spectrum with that of N-butyl-4-butylamino-1,8-naphthalimide
(FF = 0.81 in absolute ethanol), taking the area under the total
emission. The FF of 2 is 0.0026.
Cu2+ is the most notorious fluorescence quencher among
HTM ions, therefore the study of the 2–Cu2+ complex would
representatively elucidate the function of the carbonyl group.
Green crystals of 2–CuCl2 were obtained by vapor diffusion of
ether into the CH3CN solution of 2–CuCl2 (1 : 1). The single
crystal structure of 2–Cu2+ showed that the amide oxygen
cooperates with the DPA as a receptor to bind Cu2+ (Fig. 3).
The carbonyl group keeps Cu2+ away from the naphthalimide
fluorophore. Table 2 shows the selected bond distances for the
crystal of 2–Cu2+.z
The emission spectra of 2 and the effects of added Cu2+ are
shown in Fig. 4. The blue shift in emission is due to the Cu–O
bond formation, which increases the electron-withdrawing
ability of the carbonyl group. 1H-NMR analysis provides
evidence that other HTM ions are also bound to the amide
oxygen of 2 in acetonitrile, resulting in upfield shifts of the
resonance of the adjacent NH proton.13 For example, addition
of 1 equiv. of Zn2+ or Cd2+ promotes a large upfield shift
(11.72 to 9.73 and 9.49 ppm, respectively) of the resonance of
the adjacent NH proton in 2 (Fig. S12 and 13, ESIw).
Correspondingly, the absorption maximum of 2 undergoes a
blue shift from 371 nm to 348 nm upon addition of both Zn2+
and Cd2+ due to ICT (Fig. S14 and 15, ESIw).
Fig. 3 ORTEP diagram (50% probability ellipsoids) of the 2–Cu2+
complex. The crystal was grown in CH3CN–Et2O (1 : 1) with CuCl2.
Table 2 Selected bond distances (A) for the 2–Cu2+ complex
To further explore the function of the carbonyl group in 2,
fluorescence responses to HTM ions in different ratios of
water in acetonitrile were examined. With changes in water
percentages between 10 to 80%, 2 exhibited a blue shift and
increased intensity of its fluorescence response to Cu2+
(Fig. 5). As the water percentage increases beyond 80%, the
Cu(1-1)–Cl(1-1)
Cu(1-1)–O(3-1)
Cu(1-1)–N(3-1)
Cu(1-1)–N(4-1)
Cu(1-1)–N(5-1)
2.245(8)
2.316(2)
1.996(2)
2.010(2)
2.058(2)
Cu(1-2)–Cl(1-2)
Cu(1-2)–O(3-2)
Cu(1-2)–N(3-2)
Cu(1-2)–N(4-2)
Cu(1-2)–N(5-2)
2.253(8)
2.305(2)
2.007(2)
2.011(2)
2.058(2)
Fig. 4 Fluorescence spectra of 10 mM 2 in the presence of different
concentrations of Cu2+ in CH3CN (Ex: 360 nm). Inset: ratiometric
calibration curve of I430/I481 as a function of Cu2+ concentration.
Fig. 2 Fluorescence spectra of 1 in the presence of different HTM
ions in CH3CN. Excitation at 450 nm. [1] = 10 mM, [HTM] = 30 mM.
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Notes and references
z Crystal data for the 2–Cu2+ complex: C30H31Cl2CuN5O4, Mw
=
660.04, prism 0.42 ꢁ 0.21 ꢁ 0.12 mm, monoclinic P21/n (no. 14),
a = 27.4477(3), b = 6.6081(1), c = 35.2482(4) A, a = 901, b =
112.053(1)1, g = 901, V = 5925.47(13) A3, Z = 8, T = 180(2) K,
Dcalc = 1.480 g cmꢂ3, l = 0.71073 A, m = 0.962 mmꢂ1, Nonius
Kappa CCD diffractometer, 3.531 o y o 26.021, 33 837 measured
reflections, 11 470 independent (Rint = 0.0484), 8556 with I > 2s(I).
The structure was solved by direct methods (SHELXS-97) and refined
2
by least squares (SHELXL-97)16 using Chebyshev weights on Fo to
R1 = 0.040, wR2 = 0.097 [I > 2s(I)], 772 parameters. The water
hydrogen atoms were located and their positions were refined satis-
factorily, all other hydrogen atoms in calculated positions; goodness-
of-fit on F2 1.04; residual electron density 0.67 e Aꢂ3
.
1 Bioinorganic Chemistry, ed. A. X. Trautwein, Wiley-VCH,
Weinheim, 1997.
2 For selected recent examples for HTM ions: (a) T. Schwarze,
Fig. 5 Fluorescence spectra of 10 mM 2 in the presence of 30 mM
Cu2+ in acetonitrile–water with different volume ratios.
H. Muller, C. Dosche, T. Klamroth, W. Mickler, A. Kelling,
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decreasing interactions between carbonyl oxygen and Cu2+
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similar role to the carbonyl. In Kim’s report, the binding of
Cu2+ to the sulfonamide group in H2O–CH3CN (1 : 1, v/v)
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monomer emission at 375 nm. With the increase of water
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disappeared (H2O% > 70%), along with the enhancement of
the pyrene monomer emission. In the latest case, Kim et al.
exchanged the sulfonamide group with an amide group.15 The
coordination of Cu2+ by amide oxygen in CH3CN again
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HTM ions resulting in upfield shifts of the resonance of the
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In conclusion, we have presented strategies for the design
of turn-on fluorescent probes for HTM ions. The strategies
were used to design a novel probe 2 that undergoes FE in
the presence of different HTM ions and a ca. 50 nm blue shift
in emission in acetonitrile that is suitable for a ratiometric
assay. The results suggest that the carbonyl group in 2
increases the oxidation potential of the fluorophore compared
to 1, blocks HTM ions from sterically interacting with
the naphthalimide fluorophore, and that it acts as a sacrificial
donor. The observations made in this effort should serve as
the foundation for the design of new off–on probes for
HTM ions.
We thank Dr J. E. Davies for determining the crystal
structure of 2–Cu2+ and the EPSRC for financial assistance
towards the purchase of the Nonius CCD diffractometer. We
gratefully acknowledge financial support from the EPSRC,
BBSRC, MRC, Newman Trust, Herchel Smith Postdoctoral
Research Fellowship Fund, National Research Foundation of
Korea (NRF) funded by the Ministry of Education, Science
and Technology (20090083065, 20090063001).
14 H. Kim, J. Hong, A. Hong, S. Ham, J. Lee and J. S. Kim, Org.
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15 H. Jung, M. Park, D. Han, E. Kim, C. Lee, S. Ham and J. S. Kim,
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16 G. M. Sheldrick, SHELXS-97/SHELXL-97, University of Gottingen,
Germany, 1997.
¨
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Chem. Commun., 2010, 46, 1679–1681 | 1681