Cocktails of Tb3+ and Eu3+ complexes: A general platform for the design of ratiometric optical probes

Article abstract of DOI:10.1021/ja070867y

Fluorescent and luminescent reporters that signal molecular events of interest by modulating the ratio of peaks in their emission profile have advantages over reporters that simply modulate their emission intensity, since ratiometric measurement is concentration-independent and allows them to be effective in complex contexts, such as living cells or sensor microarrays. We herein describe a general platform for the design of ratiometric probes based on a heterometallic Tb³⁺/Eu³⁺ bis-lanthanide ensemble, consisting of a mixture, or "cocktail", of otherwise identical heterometalated chelates. The chelate contains an organic photon antenna that sensitizes the Tb³⁺/Eu³⁺ luminescence. The contributions of the two metals to the composite luminescence spectrum can be tuned to the same relative scale by adjusting the stoichiometry of the cocktail, allowing subtle changes in their ratio to be accurately measured. Importantly, the ratio responds to chemical and environmental changes experienced by the photon antenna, making the system an ideal platform for the design of chemical and enzymatic probes. As proofs of concept, we describe a ratiometric probe for esterase activity and a polarity-responsive ratiometric sensor.

Full text of DOI:10.1021/ja070867y

Published on Web 05/23/2007
Cocktails of Tb3+ and Eu3+ Complexes: A General Platform
for the Design of Ratiometric Optical Probes
Matthew S. Tremblay, Marlin Halim, and Dalibor Sames*
Contribution from the Department of Chemistry, Columbia UniVersity,
New York, New York 10027
Received February 6, 2007; E-mail: sames@chem.columbia.edu
Abstract: Fluorescent and luminescent reporters that signal molecular events of interest by modulating
the ratio of peaks in their emission profile have advantages over reporters that simply modulate their emission
intensity, since ratiometric measurement is concentration-independent and allows them to be effective in
complex contexts, such as living cells or sensor microarrays. We herein describe a general platform for
the design of ratiometric probes based on a heterometallic Tb3+/Eu3+ bis-lanthanide ensemble, consisting
of a mixture, or “cocktail”, of otherwise identical heterometalated chelates. The chelate contains an organic
photon antenna that sensitizes the Tb3+/Eu3+ luminescence. The contributions of the two metals to the
composite luminescence spectrum can be tuned to the same relative scale by adjusting the stoichiometry
of the cocktail, allowing subtle changes in their ratio to be accurately measured. Importantly, the ratio
responds to chemical and environmental changes experienced by the photon antenna, making the system
an ideal platform for the design of chemical and enzymatic probes. As proofs of concept, we describe a
ratiometric probe for esterase activity and a polarity-responsive ratiometric sensor.
Introduction
measurement of the eventsthis measurement is independent of
probe concentration, thereby extending its quantitative utility
The rational design of molecules that exhibit fluorescent
switching is a pursuit of great interest, since these switches can
to more complex applications, such as imaging living cells and
tissues.4 Modulation of a fluorescent reporter’s radiative lifetime
rather than the strength or position of its emission maximum is
also a viable optical reporting strategy, although it generally
requires a more complex experimental setup.5
function as reporters of chemical and enzymatic events.1-3
A
fluorescent reporter can signal an event (e.g., the binding of an
analyte or a chemical transformation) in two conceptually
different ways: (1) by increasing (fluorogenic) or decreasing
(fluorolytic) the intensity of its emission, or (2) by modulating
the ratio of peaks in its emission profile (fluoromorphic). Since
calibration of the output signal is required for quantification,
the former method yields quantitative information only in cases
where the precise concentration of the reporter is known;
otherwise, such fluorogenic/fluorolytic reporting strategies can
be relied upon only for observing qualitative trends in signal
change. On the other hand, the latter method allows ratiometric
Previous Approaches to Ratiometric Probes. In principle,
ratiometric reporting strategies can be evolved by coupling
fluorogenic or fluorolytic reporters to a second dye in one of
two scenarios: (1) the dynamic emission response of a reporter
dye is calibrated by a second dye whose emission remains
constant with respect to the parameter of interest,6 or (2) the
excited state of the responsive dye is coupled to that of a second
dye, such that their interaction is gated by the event of interest
(intramolecular quenching or resonance energy transfer).3,7
However, dual-dye systems do not offer the perfect solution,
since the first scenario requires two sets of excitation and
emission wavelengths to be monitored simultaneously, which
necessitates a more complex instrumental setup and potentially
limits the temporal resolution of consecutive data collection. If
(1) For a general review, see: (a) de Silva, A. P.; Gunaratne, H. Q. N.;
Gunnlaugsson, T.; Huxley, A. J. M.; McCoy, C. P.; Rademacher, J. T.;
Rice, T. E. Chem. ReV. 1997, 97, 1515-1566. For a recent account of
supramolecular sensors, see: (b) Anslyn, E. V. J. Org. Chem. 2007, 72,
687-699. For reviews of fluorescent enzyme activity reporters, see: (c)
Lawrence, D. S. Acc. Chem. Res. 2003, 36, 401-409. (d) Baruch, A.;
Jeffery, D. A.; Bogyo, M. Trends Cell. Biol. 2004, 14, 29-35. (e) Goddard,
J.-P.; Reymond, J.-L. Trends Biotechnol. 2004, 22, 363-370.
(2) (a) Yee, D. J.; Balsanek, V.; Sames, D. J. Am. Chem. Soc. 2004, 126, 2282-
2283. (b) Chen, G.; Yee, D. J.; Gubernator, N. G.; Sames, D. J. Am. Chem.
Soc. 2005, 127, 4544-4545. (c) Tremblay, M. S.; Sames, D. Org. Lett.
2005, 7, 2417-2420. (d) Froemming, M. K.; Sames, D. Angew. Chem.,
Int. Ed. 2006, 45, 637-642. (e) Yee, D. J.; Balsanek, V.; Bauman, D. R.;
Penning, T. M.; Sames, D. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 13304-
13309. (f) Tremblay, M. S.; Zhu, Q.; Mart´ı, A. A.; Dyer, J.; Halim, M.;
Jockusch, S.; Turro, N. J.; Sames, D. Org. Lett. 2006, 8, 2723-2726. (g)
Tremblay, M. S.; Sames, D. Chem. Commun. 2006, 4116-4118.
(3) For recent reviews of fluorescent protein-based reporters, see: (a) Zhang,
J.; Campbell, R. E.; Ting, A. Y.; Tsien, R. Y. Nat. ReV. Mol. Cell Biol.
2002, 3, 906-918. (b) Tsien, R. Y. FEBS Lett. 2005, 579, 927-932. (c)
Giepmans, B. N. G.; Adams, S. R.; Ellisman, M. H.; Tsien, R. Y. Science
2006, 312, 217-224.
(4) Demchenko, A. P. FEBS Lett. 2006, 580, 2951-2957.
(5) Excimer formation induces useful changes in fluorescence lifetimes; for
recent examples, see: (a) Mart´ı, A. A.; Li, X.; Jockusch, S.; Li, Z.;
Raveendra, B.; Kalachikov, S.; Russo, J. J.; Morozova, I.; Puthanveettil,
S. V.; Ju, J.; Turro, N. J. Nucleic Acids Res. 2006, 34, 3161-3168. (b)
Yang, C. J.; Jockusch, S.; Vicens, M.; Turro, N. J.; Tan, W. Proc. Natl.
Acad. Sci. U.S.A. 2005, 102, 17278-17283. For reviews on time-resolved
measurement of resonance energy transfer, see: (c) Wallrabe, H.; Peri-
asamy, A. Curr. Opin. Biotechnol. 2005, 16, 19-27. (d) Selvin, P. R. IEEE
J. Sel. Top. Quant. Electron. 1996, 2, 1077-1087.
(6) Woodroofe, C. C.; Lippard, S. J. J. Am Chem. Soc. 2003, 125, 11458-
11459.
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Tb3+/Eu3 Cocktail for Design of Ratiometric Probes
A R T I C L E S
the two dyes are not covalently linked,6,7d nonhomogeneous
distribution of the two dyes inside a cell may limit the spatial
dimensions over which the signal can be accurately calibrated.
Although dual-dye resonance energy transfer systems offer the
convenience of a single excitation wavelength, they still suffer
from a limitation inherent to all dual-dye systems, namely that
each dye has a different set of susceptibilities to photobleaching
and reversible environmental quenching.4 In contrast, a single
dye with responsive dual emission offers the advantage of
monitoring conversion of the reporter ratiometrically using a
single excitation wavelength.4,8 Although rare, examples of
rationally designed fluoromorphic reporters with single excita-
tion wavelengths have been reported, usually based on perturba-
tion of the equilibrium between distinctly emissive ground-state8a
or excited-state4,8b tautomers.
Design of a General Ratiometric Sensing Strategy Based
on Hetero-bis-lanthanide Ensembles. As part of a program
directed at the design and application of novel fluorescent2a-e
and luminescent lanthanide2f,g switches, we wanted to develop
responsive, ratiometric reporters based on the luminescent
lanthanide ions,9 since their sharp, long-wavelength emission
bands are ideal for biological applications, and because their
long radiative lifetimes allow background-free measurement
using time-gating.10 Because many organic chromophores are
capable of efficiently sensitizing lanthanide luminescence via
energy transfer, sensitized lanthanide complexes are a flexible
class of optical reporters and are thus an attractive platform for
the design of responsive optical switches.
The sharp lines of the lanthanide emission spectra are
essentially atomic and are consequently inert to shifts in
wavelength. As a result, a ratiometric signaling strategy based
on lanthanide luminescence is difficult to envision. Some
ratiometric sensors have been designed on the basis of modulat-
ing the relative intensities of these lines;11 however, these
changes generally result from a change in the symmetry of the
ligand sphere of a coordinatively unsaturated complexssuch
changes are difficult to predict and are known to be unreliable
in anion-rich biological contexts.2f,12 We sought a more robust
approach based on a heterometallic bis-lanthanide ensemble,
wherein luminescence from two lanthanide ions with distinct
Figure 1. Schematic representations and simplified Jabłon´ski diagrams
depicting dual-emissive bis-lanthanide ensembles sensitized by in series
(A) or in parallel (B) energy transfer originating from the excited state of
an organic photon antenna. (C) Since the in parallel mode does not involve
interaction between the metals, a heterobimetallic complex and a cocktail
of monometallic complexes will have similar optical output.
emission profiles would be combined and the relative contribu-
tion from each lanthanide varied as a function of an event of
interest. Since emission from both lanthanides will originate
from the same excited state (the organic photon antenna), their
emission ratio is independent of concentration, photobleaching
effects, and power fluctuation of the excitation source.
(7) For a recent review, see: (a) Kikuchi, K.; Takakusa, H.; Nagano, T. Trends
Anal. Chem. 2004, 23, 407-415. For recent examples, see: (b) Albers, A.
E.; Okreglak, V. S.; Chang, C. J. J. Am. Chem. Soc. 2006, 128, 9640-
9641. (c) Coskun, A.; Akkaya, E. U. J. Am. Chem. Soc. 2006, 128, 14474-
14475. (d) Bozym, R. A.; Thompson, R. B.; Stoddard, A. K.; Fierke, C.
A. ACS Chem. Biol. 2006, 1, 103-111. (e) Wichmann, O.; Wittbrodt, J.;
Schultz, C. Angew. Chem., Int. Ed. 2006, 45, 508-512. (f) Haidekker, M.
A.; Brady, T. P.; Lichlyter, D.; Theodorakis, E. A. J. Am. Chem. Soc. 2006,
128, 398-399. (g) de Silva, A. P.; Gunaratne, H. Q. N.; Gunnlaugsson,
T.; Lynch, P. L. M. New J. Chem. 1996, 20, 871-880.
Our first design was based on a heterometallic bis-lanthanide
complexsa single molecule containing discretely incorporated
Tb3+ and Eu3+ ionssthat exhibited a ratiometric Tb3+/Eu3+
emission profile as a function of solvent polarity.2g In this
system, we sensitized Tb3+ luminescence and hoped to observe
significant energy transfer13 from Tb3+ to Eu3+ and to vary the
ratio of the emission by perturbing the metal-metal energy
transfer; the overall dual energy transfer is analogous to an
electronic circuit wired in series (Figure 1A). Unfortunately,
Eu3+ emission could not be readily observed on the same relative
scale as Tb3+ luminescence, such that perturbation of their ratio
would be difficult to measure with the desired sensitivity. This
(8) (a) Chang, C. J.; Jaworski, J.; Nolan, E. M.; Sheng, M.; Lippard, S. J.
Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 1129-1134. (b) Henary, M. M.;
Wu, Y.; Fahrni, C. J. Chem. Eur. J. 2004, 10, 3015-3025.
(9) For general reviews, see: (a) Parker, D.; Dickins, R. S.; Puschmann, H.;
Crossland, C.; Howard, J. A. K. Chem. ReV. 2002, 102, 1977-2010. (b)
Bu¨nzli, J.-C. G.; Piguet, C. Chem. Soc. ReV. 2005, 34, 1048-1077. For a
review on lanthanide-based switches, see: (c) Gunnlaugsson, T.; Leonard,
J. P. Chem. Commun. 2005, 3114-3131. A key paper missing from review
9c: (d) Lee, K.; Dzubeck, V.; Latshaw, L.; Schneider, J. P. J. Am. Chem.
Soc. 2004, 126, 13616-13617. For a recent example of a lanthanide-based
protease probe, see: (e) Terai, T.; Kikuchi, K.; Iwasawa, S.-y.; Kawabe,
T.; Hirata, Y.; Urano, Y.; Nagano, T. J. Am. Chem. Soc. 2006, 128, 6938-
6946.
(10) Dickson, E. F. G.; Pollak, A.; Diamandis, E. P. J. Photochem. Photobiol.
B 1995, 27, 3-19.
(11) (a) Bretonniere, Y.; Cann, M. J.; Parker, D.; Slater, R. Org. Biomol. Chem.
2004, 2, 1624-1632. (b) Parker, D.; Yu, J. Chem. Commun. 2005, 3141-
3143. (c) Pal, R.; Parker, D. Chem. Commun. 2007, 474-476.
(12) Duimstra, J. A.; Femia, F. J.; Meade, T. J. J. Am. Chem. Soc. 2005, 127,
12847-12855.
(13) Heterobimetallic Tb3+/Eu3+ complexes have been prepared as statistical
mixtures, and energy transfer has been estimated to be efficient: Piguet,
C.; Bu¨nzli, J. -C. G. Chem. Soc. ReV. 1999, 28, 347-358.
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led us to explore a dual energy transfer analogous to a parallel
circuit, where the excited-state energy of the photon antenna
would be proportioned between the metals through direct
sensitization of both Tb3+ and Eu3+ (Figure 1B). While the series
circuit design requires the two lanthanides to exist within the
same molecule, the parallel circuit design does not; in fact, a
mixture of Eu3+ and Tb3+ complexes outfitted with the same
antenna chromophore would produce an emission response
similar to that of a bis-complex functioning in the parallel circuit
mode (Figure 1C). Since Eu3+ and Tb3+ complexes have nearly
identical behavior in solution, a “cocktail” of complexes is
expected to be homogeneous with respect to all important
propertiesssolubility, localization in a biological matrix, and
the ability to serve as an enzyme substratesand to be distin-
guishable only by their photophysical properties. The latter
rationale has been substantiated by Bornhop in the clever design
of bimodal (luminescent/magnetic) imaging agents based on
mixtures of Eu3+ and Gd3+ complexes, which are both taken
up by cells.14 Because the stoichiometry of the bis-lanthanide
ensemble is not restricted, the composition of the cocktail can
be “tuned” depending on the inherent bias of the antenna
chromophore to sensitize Tb3+ and Eu3+ emission, thus allowing
the two lanthanide profiles to be detected with comparable
sensitivity. Finally, the cocktail of luminescent complexes could
function as a ratiometric probe of physical, chemical, or
enzymatic events occurring at the antenna chromophore, since
changes in its electronic structure will be manifested in the
Figure 2. (A) Structure and numbering of the diethylenetriamine pentaacetic
acid (DTPA) conjugate of carbostyril 124 (cs124). (B) Emission spectra of
Tb:DTPA-cs124 and Eu:DTPA-cs124 normalized to the Tb3+ emission
maximum, showing the strong preferential sensitization of Tb3+ (note the
difference in scale) [25 µM DTPA-cs124, 50 µM Tb3+ (green spectrum)
or 50 µM Eu3+ (red spectrum)]. (C) A 1:1 Tb3+/Eu3+ cocktail (green
spectrum) is adjusted to bring the Eu3+ luminescence on scale with Tb3+
luminescence (black spectrum) [25 µM DTPA-cs124, 50 µM Ln3+, where
Ln3+ ) Tb3+/Eu3+ 1:1 (green spectrum) or 1:15 (black spectrum)]. All
spectra were taken in 10 mM HEPES, 100 mM NaCl (pH 7.4) at 25 °C;
excitation at 340 nm.
relative efficiencies of energy transfer to Tb3+ and Eu3+
.
To the best of our knowledge, there is only a single report
demonstrating the ratiometric sensing ability of a mixture of
Tb3+ and Eu3+ complexes, wherein binding of urate to the
lanthanide center induces selective quenching of the Tb3+
excited state (5D4).15 This example differs mechanistically and
in scope from our proposed strategy, since it relies on a specific
electronic interaction between the analyte and the metal center,
and because it involves the formation of a stable ternary complex
with the analyte.
to be detected (Figure 2C, black spectrum). Since the cocktail
is adjusted by reducing the proportion of the brighter component
(Tb3+ in this case), a consequence of adjusting the stoichiometry
is that the overall luminescence intensity is decreased.
Results and Discussion
The well-known carbostyril 124 (cs124) (Figure 2A) has the
ability to sensitize both Tb3+ and Eu3+ emission16 and serves
as a suitable scaffold for the design of antenna chromophores
that could readily sensitize a Tb3+/Eu3+ cocktail. Also, chemical
derivatization of cs124 has been shown to alter the degree to
which it sensitizes Tb3+ and Eu3+, so it should be amenable to
the design of probes exhibiting ratiometric luminescence
responses to chemical/enzymatic transformations.17 Cs124 itself
shows a strong preference to sensitize Tb3+ luminescence
(Figure 2B). Not unexpectedly, the emission spectrum of a 1:1
mixture of Tb:DTPA-cs124 and Eu:DTPA-cs124 is dominated
by Tb3+ luminescence (Figure 2C, green spectrum). However,
by simply adjusting the composition of the cocktail, the relative
luminescence intensities of the two lanthanides can be detected
with comparable sensitivity, allowing subtle changes in their
emission ratio due to environmental or chemical perturbation
Divergent Synthesis of Cs124 Derivatives. The rational
development of responsive “switches” based on a cocktail of
cs124 complexes requires understanding how functional group
changes on the cs124 scaffold affect the relative sensitization
of Tb3+ and Eu3+. In particular, we wanted to explore chemical
functionality at the 3- and 4-positions of the cs124 core, since
chemical changes at these positions produce robust fluorescent
switches in the analogous coumarin structures,2a and since
derivatization of cs124 at positions 1, 5, 6, and 8 resulted in
only modest luminescence changes.17b However, no general
method for the preparation of cs124 derivatives exists; reported
analogues have been prepared by relatively harsh methods,
which are typically low-yielding and intolerant of many
functional groups that we wish to explore.17,18 In an effort to
devise a general, divergent entry into functionalized carbostyrils,
we developed the versatile synthetic intermediate 4, which serves
as a competent coupling partner in many Pd-catalyzed cross-
coupling reactions and can be used to prepare derivatives with
(14) Manning, H. C.; Goebel, T.; Thompson, R. C.; Price, R. R.; Lee, H.;
Bornhop, D. J. Bioconjugate Chem. 2004, 15, 1488-1495.
(15) Poole, R. A.; Kielar, F.; Richardson, S. L.; Stenson, P. A.; Parker, D. Chem.
Commun. 2006, 4084-4086.
(16) Li, M.; Selvin, P. R. J. Am. Chem. Soc. 1995, 117, 8132-8138.
(17) (a) Chen, J.; Selvin, P. R. J. Photochem. Photobiol. A 2000, 135, 27-32.
(b) Ge, P.; Selvin, P. R. Bioconjugate Chem. 2004, 15, 1088-1094.
(18) Strohmeier, G. A.; Fabian, W. M. F.; Uray, G. HelV. Chim. Acta 2004, 87,
215-226.
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Scheme 1. Divergent Synthesis of 3- and 4-Substituted Carbostyrils via Triflate 4a
a Reagents and conditions: (a) allyl bromide, Na2CO3, MeCN, 80 °C, 24 h; (b) Fe0, EtOH/AcOH, 110 °C, 4 h; (c) bis(2,4,6-trichlorophenyl)malonate,
PhMe, 110 °C, 2 h; (d) PhN(SO2CF3)2, NEt3, DMF, 3 h; (e) ArB(OH)2, PdCl2(dppf), Na2CO3, DMF/H2O, 80-100 °C, 1 h; (f) ROH, Pd(OAc)2, DPPF,
DMF, CO (1 atm), 70 °C, 3 h; (g) Me3SiCCH, PdCl2(PPh3)2, CuI, NEt3, DMF, room temperature, 1 h; (h) butyl vinyl ether, Pd(OAc)2, dppp, NEt3, DMF,
80 °C, 12 h; (i) Pd(PPh3)4, N,N′-dimethylbarbituric acid, THF, 50 °C, 12 h; (j) DTPA bis-anhydride, NEt3, DMF, room temperature, 3 h; (k) K2CO3,
MeOH/CH2Cl2 (2:1), 0 °C to room temperature, 2 h; (l) 3 N HCl/AcOH (2:3), room temperature, 1 h; (m) BnN3, CuSO4, tris-benzyltriazolyl amine (TBTA),
NaAsc, t-BuOH/H2O/DMSO (2:2:1), 70 °C, 20 h; (n) NaBH4, MeOH, room temperature, 10 min; (o) n-butylamine, AcOH, 100 °C, 12 h; (p) 2 M NaOH,
EtOH, 100 °C, 4 h; (q) (i-Pr)3SiH, PdCl2(PPh3)2, NEt3, DMF, 85 °C, 24 h; (r) NBS, CH2Cl2, room temperature, 1 h; (s) PhB(OH)2, PdCl2(dppf), Na2CO3,
DMF/H2O, 80-100 °C, 1 h.
diverse substituents at the 4-position of the cs124 scaffold
(Scheme 1). Suzuki, Sonogashira, Heck, and carbonylation
reactions were used to introduce aryl/heteroaryl, alkyne, enol,
and ester functional groups, respectively.19 Elaboration of the
diallylamino quinolinones via two- or three-step procedures
involving acylation with DTPA bis-anhydride as the final step
furnished the final ligands.20 Alternatively, reductive detriflation
of 4 gave compound 20, which could be brominated and
functionalized at the 3-position. Tb3+ and Eu3+ complexes of
the DTPA conjugates were prepared by metalation with TbCl3
or EuCl3 at neutral pH.19
Photophysical Characterization of the Tb3+/Eu3+:DTPA-
Carbostyril Series. Photophysical characterization of the series
revealed that preferential Tb3+ to Eu3+ sensitization could be
altered by chemical changes at the 4-position; spectra of
representative compounds show that a wide range of lumines-
cence properties can be obtained (Figure 3). While the Tb3+
/
Figure 3. Luminescence spectra of representative 4-substituted Ln:DTPA-
cs124 derivatives, demonstrating that the inherent bias for Tb3+ or Eu3+
sensitization can be varied by chemical derivatization [25 µM substrate,
50 µM Tb3+ (green spectra) or Eu3+ (red spectra), pH 7.4 (10 mM HEPES,
100 mM NaCl), 25 °C; excitation at 340 nm].
(19) See Supporting Information for detailed synthetic procedures and charac-
terization.
(20) While the acyclic DTPA chelate is not ideal for many applications due to
its kinetic lability, it is more than sufficient for exploratory photophysical
studies and was chosen in the interest of synthetic brevity.
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Figure 4. Tb-Eu sensitization biases for the DTPA-carbostyril series. The luminescence properties of cs124 are effectively modulated by chemical
derivatization.
than 25-fold.21 Ketone 16 is also related to the 4-acetoxy
derivative 3322 by Baeyer-Villiger-type oxygen insertion;
theoretically, a 60-fold enhancement in Tb3+/Eu3+ emission ratio
would accompany such a transformation. We observed only a
modest difference in sensitization bias between 11 and 13, a
chemical switching pair related by the popular “click chemistry”
bioconjugation reaction.23 Substitution at the 3-position was not
Figure 5. Schematic representation of a responsive luminescent cocktail
investigated in any detail, since the 3-phenyl derivative 24
sensitized only Eu3+ luminescence, suggesting that even weakly
electron-withdrawing substituents in this position lower the
relevant energy donor state below the Tb3+ threshold and thus
outside the useful range for a Tb3+/Eu3+ cocktail.
undergoing a chemical/enzymatic functional group transformation (FG1 f
FG2) s the accompanying change in sensitization bias of the cocktail allows
quantitative, ratiometric measurement of conversion.
Eu3+ (or Eu3+/Tb3+) ratio is the simplest parameter for
quantifying preferential sensitization, the “sensitization bias”
(eq 1) is more descriptive and convenient for comparing the
entire series; it takes into account the difference in absolute
sensitization between Tb3+ and Eu3+, and normalizes it by the
average intensity of Tb3+ and Eu3+ luminescence.
Encouragingly, the relatively subtle differences in electronic
structure between ester 19, amide 26, and carboxylic acid 28
manifested themselves as large differences in preferential
sensitization of Tb3+ and Eu3+. Many important enzymatic
processes involve the interconversion of these three species (e.g.,
esterase- and protease-catalyzed hydrolysis, esterification, and
peptide bond formation). Worthy of mention is the fact that the
chromophores themselves (i.e., apo chelates 19, 26, and 28) do
not show significant changes in fluorescent emissionstheir
emission maxima occur at 419, 426, and 443 nm, respectively,
and the emission intensity at these maxima spans a modest range
of less than 2-fold (Figure S11, Supporting Information). Since
the fluorescence intensity change is accompanied by a shift in
wavelength, ratiometric measurement should be possible; in
practice, however, broad molecular fluorescence makes it
difficult to accurately determine emission ratios over such a
short wavelength span, particularly in microscopy applications
where the bandwidths of emission filters are typically several
tens of nanometers. This highlights an important feature of the
Tb3+/Eu3+ cocktail method: since each of the two luminescent
Tb - Eu
1/2(Tb + Eu)
sensitization bias )
(1)
where Tb and Eu represent the intergrated emission intensity
from 530 to 560 nm and from 605 to 635 nm, respectively.
Derivatization of cs124 had only subtle effects on its absorption
maximum (λmax) and molar absorptivity (ꢀ), but the sensitization
bias and average luminescence intensity were significantly
affected (Table S1, Supporting Information). Importantly, this
series of compounds demonstrates that chemical derivatization
of the carbostyril scaffold provides a wide range of dual-
emissive, supramolecular ensembles, many of which are related
through chemical transformations and can be developed into
ratiometric reporters (Figure 4).
Several luminescent switches based on functional group
transformations can be envisioned from the DTPA:cs124
derivatives and related chemical space (Figure 5). The 4-acetyl
derivative 16 and its reduction product 34 constitute an excellent
switchsthe change in the Tb3+/Eu3+ emission ratio is greater
(21) The Tb/Eu emission ratio is defined as the integrated Tb luminescence
divided by the integrated Eu luminescence; photophysical parameters for
all compounds can be found in the Supporting Information.
(22) See Supporting Information for the preparation of this compound.
(23) See a recent review and references therein: Lutz, J.-F. Angew. Chem., Int.
Ed. 2007, 46, 1018-1025.
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A R T I C L E S
Scheme 2. Synthesis of DOTAm Complexesa
a Reagents and conditions: (a) ClCH2C(O)Cl, NEt3, DMF, 0 °C, 30 min.
(b) cyclen, CHCl3/DMF (3:1), RT, 12 h. (c) BrCH2CONH2, DiPEA, DMF,
0 °C, 12 h. (d) LnCl3, EtOH/0.1M triethylammonium acetate (pH 5), RT,
12 h.
Figure 6. Enzymatic hydrolysis of DOTAm substrate 40aLn (Ln ) 1:1
Tb/Eu) catalyzed by hog liver esterase [69 µM substrate, 0.5 mg/mL enzyme,
pH 7.4, 10 mM HEPES, 25 °C; excitation at 340 nm; error bars represent
standard deviations derived from five experiments].
metals has several sharp maxima, most possessing baseline
resolution with respect to one another, it is possible to integrate
the emission intensity for each metal in a window as large as
30-40 nm without overlap between the two signals. For
example, the Tb/Eu ratio can be conveniently measured by
integrating the Tb3+[7F5] and the Eu3+[7F4] transitions (530-
560 nm and 680-710 nm, respectively). It is important to note
that the kinds of optical switches that can be developed within
this framework involve modulating the relevant electronic
excited states of the photon antenna (probably the triplet state,
T1) and cannot rely simply on the relief (or exaggeration) of a
quenching process, such as photoinduced electron transfer
(PET). Such switching mechanisms, which constitute the
majority of the fluorescent sensor literature,1a are expected to
quench both Tb3+ and Eu3+ emission without any significant
change in their ratio.9e
HPLC; the complexes coelute at a retention time that is readily
distinguishable from that of the apo chelate.26
Indeed, a cocktail of 40aTb and 40aEu allowed continuous
monitoring of enzyme-catalyzed hydrolysis (Figure 6). Specif-
ically, incubation of 40aTb/Eu (1:1) with hog liver esterase (EC
3.1.1.1) (pH 7.4, 25 °C) gave rise to a time-dependent, 4-fold
increase in the Tb3+/Eu3+ emission ratio. Uncatalyzed hydrolysis
of the substrate did not occur to any significant extent during
the course of the assay but did occur at longer time scales
(<25% after 24 h). These encouraging results demonstrated the
feasibility of using a Tb3+/Eu3+ complex cocktail to provide a
ratiometric readout of a dynamic, enzyme-catalyzed transforma-
tion in real time.
Development of a Ratiometric Esterase Activity Reporter.
We chose the ester 19/acid 28 pair to demonstrate the effective-
ness of this strategy in monitoring a dynamic, enzyme-catalyzed
chemical transformation in a continuous manner. The Tb3+/Eu3+
emission ratios are 0.35 and 1.62 for ester 19 and acid 28,
respectively,24 which should provide a comfortable 4-5-fold
contrast window in which to observe the transformation.
Compound 40aLn, an analogue of 19 in which the DTPA
chelate has been replaced by the macrocyclic DOTAm chelate,
was prepared as an enzyme substrate, since the latter possesses
superior complex stability.2f,g,20 Although DTPA and DOTAm
complexes exhibit different preferential sensitization of Tb3+
and Eu3+ due to the sensitivity of the Eu3+ emission lines to
chelate symmetry, the overall trends are the same; by adjusting
the composition of the cocktail, these differences can be
accounted for, and thus the information in Figure 4 is directly
applicable to the corresponding DOTAm complexes.25 DOTAm
analogues were prepared in a four-step sequence from the
intermediate amines described above (Scheme 2). The homo-
geneity of the DOTAm cocktails is supported by reverse-phase
Development of a Ratiometric Polarity Sensor. In addition
to responding to chemical transformations, luminescence from
the bis-lanthanide ensemble could also be used to probe physical
changes in the environment, such as polarity. As a proof of
principle, a cocktail of 40bTb and 40bEu (1:4) was subjected
to a range of solvent polarity. Titration with the nonionic
surfactant Triton X-100 induced a dramatic change in Tb3+
/
Eu3+ emission ratio, spanning a range from 10.2 to almost zero
at high surfactant concentrations (Figure 7).
Although Tb3+ and Eu3+ are by far the most studied of the
luminescent lanthanides,9 several others also exhibit sensitized
emission in the visible27 and near-infrared regions.28 A DOTAm
conjugate of the parent sensitizer cs124 (40cLn) efficiently
sensitizes Dy3+ luminescence (481 and 574 nm), which occurs
in a useful wavelength region and exhibits narrow bandwidths
characteristic of lanthanide emission. A cocktail of 40cDy and
(26) See Supporting Information for details of the HPLC experiment.
(27) (a) Petoud, S.; Cohen, S. M.; Bu¨nzli, J.-C. G.; Raymond, K. N. J. Am.
Chem. Soc. 2003, 125, 13324-13325. (b) Petoud, S.; Muller, G.; Moore,
E. G.; Xu, J.; Sokolnicki, J.; Riehl, J. P.; Le, U. N.; Cohen, S. M.; Raymond,
K. N. J. Am. Chem. Soc. 2007, 129, 77-83.
(28) (a) Faulkner, S.; Beeby, A.; Dickins, R. S.; Parker, D.; Williams, J. A. G.
J. Fluoresc. 1999, 9, 45-49. (b) Werts, M. H. V.; Woudenberg, R. H.;
Emmerink, P. G.; van Gassel, R.; Hofstraat, J. W.; Verhoeven, J. W. Angew.
Chem., Int. Ed. 2000, 39, 4542-4544. (c) Comby, S.; Imbert, D.; Chauvin,
A.-S.; Bu¨nzli, J.-C. G. Inorg. Chem. 2006, 45, 732-743.
(24) Although ester 19 does not sensitize Tb luminescence at all, residual ligand
fluorescence occuring between 530 and 560 nm causes this number to be
nonzero.
(25) Richardson, F. Chem. ReV. 1982, 82, 541-552. See Supporting Information
for further discussion of this point.
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A R T I C L E S
Tremblay et al.
excitation source fluctuation. In addition, the narrow bandwidths
and baseline resolution of the signals render their independent
measurement particularly amenable to optical microscopy. To
demonstrate the feasibility of these concepts, we have designed
a ratiometric reporter of esterase activity and a polarity-
responsive probe. The photophysical mechanisms underlying
the observed ratiometric transformations require more detailed
study, but they most likely result from a combination of two
factors: alteration of the energy of the antenna’s donor state,
which in turn affects the partitioning of energy transfer between
the two metals, and the differential sensitivity of the lanthanide
excited states to environmental changes. One limitation of the
system is the relatively narrow energy range required for
sensitization of both Tb3+ and Eu3+sthe allowable electronic
changes that a chromophore can experience and stay within this
range may restrict the types of chemical and enzymatic
transformations for which reporting cocktails can be developed.
However, the success of 40aTb/Eu in monitoring enzyme-
catalyzed ester hydrolysis suggests that, within this range, the
lanthanide cocktail strategy may provide greater sensitivity to
subtle electronic changes. Another limitation is the ultraviolet
excitation required by these chromophores; light of this wave-
length can be damaging to biomatter and is not ideal for many
biological applications. Nevertheless, two-photon excitation of
chromophores capable of sensitizing Tb3+ and Eu3+ may be a
viable way to address this challenge.30
Figure 7. Solvent-polarity-dependent, ratiometric luminescence response
of 40bLn (Ln ) 1:4 Tb/Eu) [50 µM, deionized H2O, titrated with Triton
X-100 (100 mg/mL), 25 °C; excitation at 340 nm; error bars represent
standard deviations derived from three experiments].
Figure 8. No change in the luminescence of a cocktail of 40cDy and 40cEu
(1:9) is observed upon titration with Triton X-100 [50 µM, deionized H2O,
titrated with Triton X-100 (100 mg/mL), 25 °C; excitation at 340 nm; error
bars represent standard deviations derived from three experiments].
40cEu (1:9) demonstrates the complementarity and minimal
overlap of the two lanthanide spectra (Figure 8). Interestingly,
the Dy3+/Eu3+ emission ratio was inert to polarity changes
induced by the addition of nonionic surfactant.29 The reduced
sensitivity of Dy3+ to environmental changes suggests a potential
role for it as a replacement for Tb3+ in applications of the
cocktail strategy where fluctuating Tb3+ emission due to
environmental changes may convolute the ability to read out a
chemical or enzymatic event. Also, since the Dy3+ band centered
at 574 nm occurs conveniently between the dominant Tb3+ and
Eu3+ bands (centered at 545 and 613 nm, respectively), the
possibility exists for simultaneous multi-variable readout using
a tris-lanthanide cocktail consisting of all three ions.
Experimental Section
Chemical Synthesis. Detailed synthetic procedures and characteriza-
tion data for all DTPA and DOTAm conjugates can be found in the
Supporting Information. Purification of final chelates was done by
preparative reverse-phase HPLC using increasing linear gradients of
90% MeCN/H2O in H2O (0.1% trifluoroacetic acid).
Metalation of Chelates. Metalation of DTPA chelates (7a-n, 11,
13, 14, 16, 19, 24, 26, 28, 34) was carried out by incubating the chelate
(25 µM in 10 mM HEPES, 100 mM NaCl, pH 7.4) with the appropriate
LnCl3‚6H2O salt (50 µM) for 2 h. These solutions were used directly
for exploratory photophysical measurements. Complexes used for
enzyme and polarity assays (40a-cLn) were prepared by metallating
the DOTAm chelates (1 equiv) with the appropriate LnCl3‚6H2O salt
(4 equiv) in 1:1 EtOH/0.1 M triethylammonium acetate buffer (pH ∼5).
After 12 h at room temperature, the complexes were purified by
preparative HPLC as described above, giving white solids after
lyophilization. All complexes were pure by HPLC and gave satisfactory
mass spectral data (see Supporting Information).
Conclusion
We have described a general strategy for the rational design
of ratiometric, luminescent probes based on a heterometallic
bis-lanthanide platform. The cocktail of Eu3+ and Tb3+ (or
Dy3+) complexes exhibits a highly structured, information-rich
composite luminescence spectrum with several sharp, baseline-
resolved emission lines that blanket the visible region from 470
to 705 nm. The relative intensities of the emission lines can be
tuned by adjusting the stoichiometry of the cocktail without
disrupting its physical homogeneity, since the lanthanide chelates
have nearly identical properties in solution. Since the lumines-
cence of the mixture is sensitized by a single antenna, changes
in its electronic structure can manifest themselves as changes
in the relative ratio of luminescence from each metal. When
these changes are coupled to chemical/enzymatic processes or
environmental changes, a ratiometric reporting strategy emerges
that is independent of probe concentration and s as a result of
having a single excitation wavelength s is independent of
Photophysical Characterization of the DTPA-Carbostyril Series.
Molar extinction coefficients were determined in 10 mM HEPES, 100
mM NaCl, pH 7.4 buffer using five different concentrations in the range
20-100 µM from two independently weighed stock solutions. Emission
spectra were measured following excitation at 340 nm for all
compounds. Sensitization bias was determined by eq 1 using the
integrated Tb3+ (530-560 nm) and Eu3+ emission (605-635 nm); data
were processed using Microsoft Excel. Complete photophysical data
for the series can be found in the Supporting Information, Table S1.
Esterase Assay. A cocktail of 40aTb and 40aEu (1:1) (69 µM) was
treated with hog liver esterase (EC 3.1.1.1, Fluka catalog no. 46058,
0.5 mg/mL) in pH 7.4 HEPES (10 mM) at 25 °C, and emission spectra
were taken at various time points (Figure 6). Samples were excited at
340 nm, and emission was measured from 400 to 750 nm. The Tb/Eu
(30) (a) Werts, M. H. V.; Nerambourg, N.; Pe´le´gry, D.; Grand, Y. L.; Blanchard-
Desce, M. Photochem. Photobiol. Sci. 2005, 4, 531-538. (b) Piszczek,
G.; Maliwal, B. P.; Gryczynksi, I.; Dattelbaum, J.; Lakowicz, J. R. J.
Fluoresc. 2001, 2, 101-107.
(29) This resistance to polarity-induced luminescence change is not a property
of the cs124 chromophore; Tb:cs124 complexes exhibit polarity-dependent
emission similar to that of 40bTb.
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+
Tb3+/Eu3 Cocktail for Design of Ratiometric Probes
A R T I C L E S
emission ratio was calculated using the integrated emission intensity
from 535 to 555 nm for Tb and the integrated emission intensity from
680 to 705 nm for Eu. Uncatalyzed hydrolysis of the substrate does
not occur to any significant extent during the course of the assay but
does occur at longer time scales (∼25% after 24 h). Compound 40aLn
is not an optimized substrate for this enzyme; due to the relatively
high concentration of enzyme required to effect this transformation over
the course of 4-5 h, detailed kinetic parameters were not measured.
as a stock solution (100 mg/mL in H2O) and mixed thoroughly before
measurement (Figure 7). The same procedure was used for a cocktail
of 40cDy and 40cEu (1:9).
Acknowledgment. The authors thank the G. Harold & Leila
Y. Mathers Charitable Foundation for generous financial sup-
port. M.H. is the recipient of the Novartis Graduate Fellowship
in Organic Chemistry.
Supporting Information Available: Synthetic procedures and
characterization for all compounds and additional photophysical
data. This material is available free of charge via the Internet
at http://pubs.acs.org.
Polarity Assay. The Tb/Eu emission ratio (defined as above for the
esterase assay) from a cocktail of 40bTb and 40bEu (1:4) (50 µM)
was measured at a variety of concentrations of Triton X-100 (poly-
ethylene glycol tert-octylphenyl ether), which was added incrementally
JA070867Y
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