Design of emission ratiometric metal-ion sensors with enhanced two-photon cross section and brightness

Article abstract of DOI:10.1021/ja073240o

To develop metal-responsive fluorescence probes with optimized nonlinear optical properties for two-photon excitation fluorescence microscopy (TPEM), we investigated the utility of donor-acceptor architectures where the metal-ion coordinates to the acceptor rather than donor site of the fluorophore. Zn(II) binding to such a probe consisting of a tris(picolyl)amine-substituted acceptor site connected with an anisole donor moiety and an oxazole central core resulted in a significant increase of the two-photon absorption cross section (δ) and fluorescence brightness (ηδ). The fluorophore showed also a strong shift of the emission peak upon saturation with Zn(II), rendering the proposed approach suitable for two-photon excited emission ratiometric sensing. Copyright

Full text of DOI:10.1021/ja073240o

Published on Web 09/11/2007
Design of Emission Ratiometric Metal-Ion Sensors with Enhanced
Two-Photon Cross Section and Brightness
S. Sumalekshmy, Maged M. Henary, Nisan Siegel, PaDreyia V. Lawson, Yonggang Wu,
Karin Schmidt, Jean-Luc Bre´das,* Joseph W. Perry,* and Christoph J. Fahrni*
School of Chemistry and Biochemistry, Petit Institute for Bioengineering and Bioscience, and Center for Organic
Photonics and Electronics, Georgia Institute of Technology, 901 Atlantic DriVe, Atlanta, Georgia 30332
Received May 7, 2007; E-mail: fahrni@chemistry.gatech.edu
Scheme 1
Two-photon excitation fluorescence microscopy (TPEM) has
rapidly evolved into a widely used tool in biological and biomedical
research.1 Compared to traditional fluorescence microscopy, TPEM
offers intrinsic 3D resolution combined with reduced phototoxicity,
increased specimen penetration, and negligible background fluo-
rescence. At present, most fluorophores used as labels or sensor
platforms in TPEM have been adopted from linear microscopy and
are not optimized for two-photon excitation.2 Notably, the fluo-
rescence brightness (ηδ), defined by the product of TPA cross
section (δ) and emission quantum yield (η), is typically low due to
a modest δ.3 The development of new TPEM-optimized fluoro-
phores is particularly vital in the context of biological metal-ion
sensing since most of currently available ratiometric sensors,
including the widely used dyes fura-2 and indo-1,4 exhibit a low
brightness that decreases even further upon cation binding. In
addition, the majority of ratiometric metal-ion sensors offer only a
large shift of the excitation peak but not emission energy. If only
a single two-photon excitation source is on hand, such sensors are
not suitable for dynamic ratiometric TPEM imaging with temporal
resolution. In this communication, we address these problems with
a molecular design approach that yields both an increase in δ and
a shift of the peak emission energy upon metal-ion binding in a
polar environment.
Molecular design strategies of organic molecules with large δ
are well-established.5,6 In general, the magnitude of δ increases with
an increasing degree of intramolecular charge transfer (ICT) upon
excitation. For example, centrosymmetric fluorophore architectures
consisting of a conjugated linear π-system with an acceptor moiety
sandwiched between two electron donors (D-π-A-π-D) exhibit
exceptionally large δ values.6 However, in water, the highly
polarized excited state gives rise to enhanced solvent-solute
interactions, which in turn leads to a reduced δ, more efficient
nonradiative deactivation, and thus to a drastically reduced bright-
ness.7 The centrosymmetric architecture poses additional challenges
for the design of metal-ion sensors: (1) the interpretation of the
sensor response is complicated due to the presence of two metal
binding sites (Scheme 1a); (2) metal binding induces a reduction
of ICT which in turn results in a smaller δ accompanied by a
strongly blue-shifted peak excitation energy; and (3) partial
decomplexation or ejection of the metal cation in the excited state
is presumably responsible for typically small emission shifts that
are not suitable for ratiometric sensing.8 Here, we propose to
overcome these problems by using a simplified D-A9 motif where
the metal-ion binds to the acceptor rather than donor site (Scheme
1b). Besides eliminating the second binding site, such an arrange-
ment should yield an increase rather than decrease of ICT upon
excitation. As a consequence, metal-ion binding is expected to result
in an increased δ, enhanced fluorescence brightness, and a red-
shifted peak emission suitable for ratiometric measurements.
Table 1. Computed One and Two-Photon Photophysical
Properties of the Free and Zn(II) Complexes of Fluorophores 1
and 2a
1
[1-Zn]SO4
2
[2-Zn]SO4
Ege (eV), λ (nm)
3.54, 350 3.30, 376 3.53, 351 3.32, 373
pωmax (eV), λmax(nm) 1.77, 701 1.65, 752 1.76, 703 1.66, 748
δ
max (GM)b
ge (D)
38
62
41
84
M
7.6
7.9
7.5
7.7
a Based on geometries optimized at the B3LYP/6-31G* level of theory
(see Supporting Information for details). b Unit Go¨ppert-Mayer (1 GM )
10-50 cm4s/photon).
Quantum chemical calculations have proven to be a reliable tool
for rationalizing structure-property relationships of two-photon
absorbing fluorophores.5,6,10 To evaluate the proposed design, we
calculated δ based on the well-established methodology combining
the sum-over-states (SOS) perturbative approach with highly
correlated MRD-CI (multireference determinant-configuration
interaction) calculations of the excited states based on the INDO
Hamiltonian.5,10 Given our interest in Zn(II)-selective fluorescent
probes,11 we combined a tris(picolyl) metal-chelating unit12 with
an anisole donor linked through a rigid oxazole core13 (fluorophore
1). The calculations indicate that Zn(II) coordination to the acceptor
site should increase the gas-phase TPA cross section of 1 by almost
a factor of 2, provide a significant red shift of the two-photon peak
absorption energy from 1.77 to 1.65 eV, and enhance the oscillator
strengths (Table 1).
Encouraged by these results, we synthesized 1 and studied its
properties. Spectrochemical titrations in methanol or water as
solvent revealed that 1 coordinated to Zn(II) with a 2:1 rather than
1:1 stoichiometry, an unexpected finding that was further confirmed
by Job’s method of continuous variation (Supporting Information).
To avoid misinterpretations of the photophysical properties due to
the presence of multiple coordination equilibria, we investigated
fluorophore 2 with a modified chelating moiety containing two
weaker coordinating 6-fluoropyridine ligands. As evident from
Table 1, the photophysical properties of 1 were predicted to remain
essentially unaltered upon fluorine substitution. Consistent with a
single, well-defined coordination equilibrium, UV-vis titration of
9
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J. AM. CHEM. SOC. 2007, 129, 11888-11889
10.1021/ja073240o CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
considerable emissive quantum yield of both the free and Zn(II)-
bound fluorophores (Table 2), the fluorescence brightness δη
increased 5-fold from 11 to 55 GM, thus exceeding the brightness
of many fluorophores widely used in biology, including fluorescein,
BODIPY, DAPI, or GFP.3,4 Compared to fluorescein, 2 showed
also improved photostability (Supporting Information). Most im-
portantly, unlike fluorophores with a D-π-A-π-D architecture,
fluorescence emission of 2 is not reduced but substantially enhanced
upon Zn(II) coordination. It is worthwhile noting that at wavelengths
above 800 nm δ of 2 is negligible in the absence but strongly
enhanced in the presence of Zn(II), thus offering a strong turn-on
response (>10 000-fold increase) of the fluorescence emission in
this spectral range.
Figure 1. Absorption (left) and fluorescence emission (right) spectra for
the titration of fluorophore 2 (21 µM) with Zn(OTf)2 in MeOH.
In conclusion, we have demonstrated that metal-ion coordination
to the acceptor site of fluorophores with a D-A architecture results
not only in a significant increase of the TPA cross section and
brightness but also a substantial shift of the peak emission energy
suitable for ratiometric sensing. This approach is not limited to the
design of Zn(II) sensors but should be applicable to a broad range
of donor-acceptor fluorophores modified with tailored chelating
sites for selective metal-ion sensing.
Acknowledgment. Financial support by NIH (DK68096), NSF
CDMITR STC (DMR-0120967), ONR MURI (N00014-03-1-0793),
and NSF CRIF CHE-0443564 is gratefully acknowledged.
Figure 2. Two-photon excitation spectra of fluorophore 2 (100 µM solution
in MeOH) in the absence (b) and presence (O) of Zn(II).
Supporting Information Available: Procedures for the synthesis
1
of 1 and 2, complexation studies, H NMR titrations, and a summary
Table 2. Photophysical Dataa for Fluorophore 2 and [2-Zn]2+
of the computational results (Cartesian atomic coordinates for the
optimized geometries and the photophysical data). This material is
available free of charge via the Internet at http://pubs.acs.org.
+
2
[2-Zn]2
absorption λmax (nm)
emission λmax (nm)
quantum yieldd
338 (2.15)b
441c
0.35
11
31 (690)
362 (2.27)b
497c
0.71
55
77 (730)
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