Luminescent lanthanide complexes with analyte-triggered antenna formation

Article abstract of DOI:10.1021/ja3004045

A new strategy for accessing analyte-responsive luminescent probes is presented. The lanthanide luminescence of Eu and Tb centers is switched on by the analyte-triggered formation of a sensitizing antenna from a nonsensitizing caged precursor. As the cage can be freely varied, an array of probes for different analytes (Pd^0/2+, H₂O₂, F ^-, β-galactosidase) can be created from the same core structure. The probe design affords nanomolar to micromolar detection limits, provides the capability to detect two analytes in parallel, and can be utilized to monitor enzymatic activity in live cells.

Full text of DOI:10.1021/ja3004045

Communication
pubs.acs.org/JACS
Luminescent Lanthanide Complexes with Analyte-Triggered
Antenna Formation
Elias Pershagen,‡ Johan Nordholm,† and K. Eszter Borbas*,‡,§
†
‡Department of Organic Chemistry and Department of Biochemistry and Biophysics, Arrhenius Laboratory, Stockholm University,
106 91 Stockholm, Sweden
S
* Supporting Information
importance,7 these species also cover an unprecedented breadth
of analyte types.
We envisioned that a nonsensitizing antenna precursor such
as the caged coumarin in Ln1 could be built into a Ln complex
framework (Figure 1). Uncaging by the analyte triggers a
ABSTRACT: A new strategy for accessing analyte-
responsive luminescent probes is presented. The lantha-
nide luminescence of Eu and Tb centers is switched on by
the analyte-triggered formation of a sensitizing antenna
from a nonsensitizing caged precursor. As the cage can be
freely varied, an array of probes for different analytes
(Pd0/2+, H2O2, F−, β-galactosidase) can be created from
the same core structure. The probe design affords
nanomolar to micromolar detection limits, provides the
capability to detect two analytes in parallel, and can be
utilized to monitor enzymatic activity in live cells.
luorescence spectroscopy is a sensitive, minimally invasive
F
technique for the monitoring of biologically relevant
species and processes and provides a simple, low-cost
alternative to established techniques (e.g., atomic absorption/
emission spectroscopy) for the detection of environmental
contaminants. Responsive probes based on luminescent
lanthanides (Ln), which combine superior emission properties1
with versatility, are made possible by placing Ln sensitization by
the light-harvesting antenna under analyte control.2 Ln probes
1
to measure citrate and lactate in biological fluids and pH, O2,
and NO in cells have been reported.3 Despite these successes,
the development of new responsive Ln probes is still a major
undertaking. The current strategies for creating new probes are
often tedious and/or are applicable to only a single analyte
without the possibility of extension to additional ones.2,4 The
narrow, nonoverlapping Ln emissions enable ratiometric
imaging with mixtures of Eu and Tb complexes5 and the
detection of multiple luminescent labels.6 However, to the best
of our knowledge, the simultaneous detection of two analytes
with responsive Ln probes has not been reported, possibly
reflecting the difficulties of constructing two noninterfering
probes using established methods.
Here we present a Ln probe design that (1) is simple to
synthesize, (2) gives a turn-on emission over zero background,
(3) is selective for a single analyte over potentially competing
ones, and (4) can easily be adapted to detect a wide array of
different analytes by only a predictable modification of the core
structure. This design yields significant improvements over
previously reported H2O2-responsive Ln probes and has
enabled the creation of new Ln probes for analytes for which
only organic-fluorophore-based probes have been reported
(Pd0/2+, F−, β-galactosidase). In addition to being valid targets
in their own right because of their environmental and biological
Figure 1. Analyte-triggered antenna formation.
reaction that forms the sensitizing antenna in situ (a coumarin
in Ln2; Figure 1). As sensitization is dependent on a
chemoselective reaction as opposed to a binding event, the
development of Ln probes for species that are difficult to detect
with supramolecular probes (e.g., several transition metals,
Received: January 13, 2012
Published: February 16, 2012
© 2012 American Chemical Society
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neutral molecules) is possible.8 The same architecture can carry
different caging groups; thus, a single molecular design can be
used for diverse analytes. Coupling this with coumarins, which
are known sensitizers of both Tb and Eu,9 further enhances the
application of the design by enabling the independent
monitoring of two analytes with two probes that possess two
different caging groups. Analyte-mediated introduction of the
antenna to obtain a turn-on response over zero background has
been used before to detect Cu(I)4a and OH•.4d However, the
detection mechanisms of those probes are restricted to their
respective analytes. Furthermore, their bimolecular nature
precludes intracellular applications, as this would require
efficient colocalization of the Ln complex, antenna, and analyte.
Benzylboronic acid-caged10 Ln1a (detects H2O2), allyl-
caged11 Ln1b (detects Pd0/2+), TIPS-caged12 Eu1c (detects
F−), and galactose-caged Eu1d (detects β-galactosidase) were
synthesized by the short route shown in Scheme 1. Caging
Caged Ln1a−d were essentially nonemissive when excited at
356 nm, while Eu2 and Tb2 displayed intense Ln-centered
luminescence (>80-fold larger emission at 595 nm for Eu2
relative to Eu1b; ΦEu2 = 0.31%). Exposure of Ln1a−d to their
respective analytes switched on the Ln emission (Figures S1−
S5 in the SI). The identity of the emissive product was
confirmed to be Ln2 by high-resolution electrospray ionization
mass spectrometry and UV−vis and fluorescence spectroscopy.
Uncaging and coumarin formation were seen when the 5a−c
1
→ 7 reaction (Scheme 2) was followed by H NMR analysis
Scheme 2
(Figures S6 and S7). The number of Ln-bound water molecules
in Eu2 was measured16 to be ∼1 (Table S1 in the SI), in
accordance with the eight-coordinate macrocyclic ligand
structure.
a
Scheme 1. Synthesis of Ln Probes
We investigated the analyte-triggered reactions Ln1a−d →
Ln2. Time-resolved luminescence titrations of Eu1a and Tb1a
showed that the probe response was linear in the 0−200 μM
[H2O2] range. H2O2 concentrations as low as 1 μM could be
detected at pH 7 at ambient temperature (Figure 2), which
Figure 2. Time-resolved luminescence titration of Eu1a with H2O2
(10 μM Eu1a, pH 7 HEPES buffer, 1 h, r.t., λex = 356 nm, λem = 615
nm, 50 μs delay, 1050 μs sample window). Inset: Eu luminescence at
[H2O2] = 0, 1, and 2 μM (1.5 h, r.t., λem = 594 nm).
a
Conditions: (i) R−Br, K2CO3, CH3CN (4a, 4b, 4d) or TIPS-Cl,
DBU, CH2Cl2 (4c); (ii) diethyl malonate, piperidine (cat.), 3 Å
molecular sieves, CH3CN (then for 4d, K2CO3, MeOH); (iii) CuSO4,
NaAsc, TBTA, 2:1:1 H2O/tBuOH/THF.
bodes well for potential biological applications. H2O2 has been
implicated in the emergence of pathologies such as cancer and
neurodegenerative diseases and is also a mediator of signal
transduction, making its detection at low micromolar
concentrations an important goal.7a,b,17 The performance of
Eu1a is comparable to those of the best organic-chromophore-
based fluorescent probes18 and superior to those of known Ln-
based H2O2 probes, with higher sensitivity and a larger turn-on
response (>30-fold, 30 min, 1 mM H2O2).19
groups were attached either directly to the phenolic oxygen or
via a 4-benzyloxy linker; in the latter case, initial uncaging was
followed by quinone−methide13 elimination [Scheme S1 in the
Supporting Information (SI)]. Aldehyde 314 was O-alkylated
with the required bromides or silylated with TIPS-Cl, yielding
4a−d, which in turn were treated with excess diethyl malonate
in the presence of 3 Å molecular sieves and catalytic piperidine
to afford 5a−d in good to excellent yields. Importantly, the
caged antennae 5a−d were combined with Ln6 in the final step
in a Cu(I)-catalyzed cycloaddition.15 This reaction is tolerant of
caging groups that could succumb to the harsh conditions
traditionally employed for ligand synthesis and complexation
(i.e., acidic/basic ester hydrolysis, prolonged heating with Ln
salts).
We found that Eu1b could detect Pd0 [using Pd(PPh3)4 as a
source] and Pd2+ in the presence of PPh3 (Figure 3a and Figure
S8). Eu1b was selective for Pd0 over a range of metal cations
(Figure 3b), with only π-philic20 Pt2+ reacting with Eu1b
(∼18% of the response observed with Pd0). Detection of Pd in
the presence of these cations revealed that only Au3+ interfered
with Eu2 formation (Figure S9), likely because Au3+ is a strong
oxidant.21
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Figure 4. Monitoring of de novo β-galactosidase synthesis with Eu1d
(10 μM Eu1d, λex = 356 nm, λem = 590 nm, 37 °C).
nontoxic to the cells as well, since neither Eu2 nor Eu1d
interfered with bacterial growth up to 25 μM (Figure S13).
These results highlight the potential application of these
compounds for monitoring of cellular processes, as Eu1d must
be able to enter the cells in order to undergo uncaging by
intracellular β-galactosidase (Figure S14). Eu1d uptake could
potentially take place via a galactose-dependent transport
mechanism23 or passive diffusion through the cell membrane.3c
The bulk of Eu2 formed from Eu1d was localized in the
extracellular milieu. As β-galactosidase was intracellular
throughout the experiment (Figure S14), the participation of
one or more of the bacterial efflux systems24 in the clearance of
Eu2 is possible.
Figure 3. (a) Evaluation of Pd sources. (b) Selectivity of Eu1b for Pd0
over metal cations (Zn2+, Ru3+, Rh+, Pt2+, Ni2+, Mn2+, Mg2+, Li+, K+,
Fe3+, Fe2+, Cu2+, Co2+, Au3+, Au+, and Ag+ left to right). (c) Titration
curve for Eu1b with Pd0 (10 μM Eu1b, CH3CN:pH 10 borate buffer
(1:1), 10 min, r.t., λex = 356 nm, λem = 699 nm).
Finally, we wanted to establish whether our probes could
simultaneously detect two analytes in parallel. To address this,
we incubated the H2O2 probe Tb1a with 50 or 200 μM H2O2
in the presence of the fluoride probe Eu1c and 200 μM TBAF.
With increasing [H2O2], a concentration-dependent increase in
the Tb emission bands (545 and 490 nm) was observed. This
increase was similar to that for the sample lacking TBAF.
Control experiments with TBAF but without H2O2 gave only
background signal levels. As the 592 and 614 nm Eu emissions
overlap with the Tb 5D4 → 7FJ bands (J = 4, 586 nm; J = 3, 621
nm), we monitored the 5D0 → 7F3 (655 nm) Eu band, to which
the Tb contribution is negligible.1,2 The intensity of this
emission band was [H2O2]-independent and similar to that
observed in a control experiment with Eu1c and [F−] = 200
μM (Figure 5).
Fluorescence titration of Eu1b with Pd(OAc)2/PPh3 showed
that [Pd] as low as 80 nM elicited Eu emission within 10 min at
ambient temperature, with a linear response in the 0−8 μM
range (Figure 3c). We expect that this remarkable performance,
which is comparable to those of the best organic-chromophore-
based probes,11,22 could be further improved by additional
optimization of the detection conditions. Pd is a known
environmental contaminant and Pd-catalyzed reactions are
popular for drug synthesis, making Pd detection of considerable
interest.7c,d
In the presence of fluoride ions, Eu1c provided an increase in
Eu emission that was concentration-dependent over the 0−200
μM range, with a detection limit of ∼2 μM in DMSO (Figure
S10). Taken together, these results show that our probe design
can be readily applied to the sensitive detection of a broad
variety of small-molecular weight species.
Analyte-triggered antenna formation also enables the
development of turn-on Ln probes for enzymatic activity.
Exposure of Eu1d to purified β-galactosidase yielded intense Eu
emission, a result that was reproduced in lacZ+ bacterial cell
lysates but not the control lacZ− bacterial cell lysates (Figures
S11 and S12). More impressively, β-galactosidase activity could
be monitored in real time during live cell growth. The addition
of 10 μM Eu1d to the culture medium of lacZ+ bacteria
constitutively expressing β-galactosidase, but not the addition
to lacZ− bacteria, resulted in an intense Eu-centered emission
within ∼5 min that increased until reaching a plateau at ∼90
min (Figure 4). Having established that the time scale of Eu1d
uptake and turnover was ∼5 min, it was reasonable to interpret
the gradual increase in Eu emission as a sign of de novo β-
galactosidase synthesis. These compounds were apparently
In conclusion, a new design strategy for responsive Ln-based
luminescent probes that opens the door to probes with very
low background, ease of synthesis, versatility, and high analyte
specificity has been developed. Current work is focused on the
development of probes for additional analytes and optimization
of Ln sensitization by shortening the Ln−antenna distance and
thus increasing the quantum yield.25 We are also exploring
alternative antenna-forming reactions.26 We have demonstrated
the versatility of our design by developing probes for the
detection of chemically quite distinct species as well as
enzymatic activity. In view of the abundance of chemoselective
reactions mediated by low-molecular-weight species (e.g., Hg2+,
27a−c
•−
H2S, O2
)
and enzymes (e.g., hydrolases, oxidoreducta-
ses)27d−g that reveal a phenolic OH group, we are confident
that this design will prove to be exceptionally broad and useful
in many new areas as well as those where Ln emission
regulation is required.
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ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental procedures, characterization of all new com-
pounds, Figures S1−14 and Table S1. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
eszter.borbas@kemi.uu.se
■
(18) (a) Miller, E. W.; Albers, A. E.; Pralle, A.; Isacoff, E. Y.; Chang,
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Present Address
§Department of Chemistry - BMC, Uppsala University, 751 23
Uppsala, Sweden.
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J. Chem. Commun. 2010, 46, 7510.
(20) Yamamoto, Y. J. Org. Chem. 2007, 72, 7817.
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
We thank the Swedish Research Council and the Department
of Organic Chemistry of Stockholm University for funding, Dr.
James Bruce (The Open University, Milton Keynes, U.K.) for
access to a fluorimeter, Prof. Gunnar von Heijne for access to a
plate reader, and Dr. Robert Daniels for bacteria (Department
of Biochemistry and Biophysics, Stockholm University). We
thank Dr. James Bruce, Dr. Robert Daniels, Prof. Jonathan
Lindsey, Helena Lundberg, Dr. Luke Pilarski, and Dr. Timofei
Privalov for pertinent discussions.
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