Ratiometric and intensity-based zinc sensors built on rhodol and rhodamine platforms

Article abstract of DOI:10.1021/ic101513a

A xanthene-forming condensation reaction yields rhodol and rhodamine dyes carrying a zinc-binding ligand that includes the aniline-type nitrogen donor of the fluorophores. Upon zinc coordination in neutral aqueous solution, rhodol RF3 behaves as a ratiometric sensor, and rhodamine RA1 acts as a turn-off intensitybased indicator. Both fluorescent compounds bind the divalent zinc cation with micromolar affinity.

Full text of DOI:10.1021/ic101513a

Inorg. Chem. 2010, 49, 9113–9115 9113
DOI: 10.1021/ic101513a
Ratiometric and Intensity-Based Zinc Sensors Built on Rhodol and
Rhodamine Platforms
Elisa Tomat and Stephen J. Lippard*
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
Received July 28, 2010
A xanthene-forming condensation reaction yields rhodol and
rhodamine dyes carrying a zinc-binding ligand that includes the
aniline-type nitrogen donor of the fluorophores. Upon zinc coordi-
nation in neutral aqueous solution, rhodol RF3 behaves as a
ratiometric sensor, and rhodamine RA1 acts as a turn-off intensity-
based indicator. Both fluorescent compounds bind the divalent zinc
cation with micromolar affinity.
sities at two chosen wavelengths corresponds to a specific
analyte concentration, which can be calculated from micro-
scopy data in dual excitation or dual emission experiments.
When compared to intensity-based systems, which respond
to zinc binding with a change in emission intensity, ratio-
metric sensors are preferred in bioimaging applications
because their detection is less sensitive to dye concentration,
sample thickness, and photobleaching.
For the construction of a novel ratiometric indicator, we
reexamined the asymmetric platform of rhodol (also known
as rhodafluor) dyes. These rhodamine-fluorescein hybrid
molecules retain several desirable properties of the parent
compounds, namely water solubility, brightness, and visible-
light excitability.15 The subject of recent synthetic investi-
gations,16,17 rhodols have been employed in metal ion
sensing. In order to elicit a ratiometric response, the coordi-
nating analyte should interact directly with the π-system
of the fluorophore, thereby affecting the energy levels that
are responsible for emissive transition(s). The nitrogen
donor of rhodol platforms has been incorporated in well-
established chelators, such as 1,2-bis(o-aminophenoxy)ethane-
N,N,N0,N0-tetracetic acid (BAPTA),18 1,4,7,10-tetraazacy-
clododecane (cyclen),19 and di(2-picolyl)amine (DPA).19
Whereas the BAPTA systems are ratiometric calcium sen-
sors, the DPA- and cyclen-based sensors (RF1 and RF2,
respectively, Chart 1) show little or no affinity for zinc and do
not exhibit ratiometric behavior.
In the present study, we modified the structure of RF1 to
increase its zinc affinity and to engineer a zinc-induced shift
of emission wavelength. A carboxylate moiety, negatively
charged in neutral aqueous solution, was introduced on the
DPA unit to increase zinc-binding affinity and to influence
the charge distribution of the sensor candidate RF3
(Chart 1). Coulombic interactions with the negative charge
on the carboxylate group were expected to stabilize a partial
positive charge on the nitrogen donor of the rhodol scaffold.
Bioinorganic research continues to unveil the critical and
often multifaceted roles of loosely bound metal ions, parti-
cularly late elements of the first transition series, in human
health.1-3 Discoveries about the (patho)physiology of these
metal ions both stimulate and benefit from research on
imaging techniques for biological specimens.4-7 In this con-
text, fluorescent probes for the spectroscopically silent zinc
ion contribute to the study of mobile zinc signals in numerous
biological settings,8-10 including the central nervous11 and
immune systems.12
Considerable effort has been devoted in recent years to the
development of fluorescent indicators for ratiometric detec-
tion of biological zinc.8,13,14 Probes of this type respond to
zinc coordination with a shift of their emission and/or
absorption profiles. As such, each ratio of measured inten-
*To whom correspondence should be addressed. E-mail: lippard@
mit.edu.
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r
2010 American Chemical Society
Published on Web 09/22/2010
pubs.acs.org/IC

9114 Inorganic Chemistry, Vol. 49, No. 20, 2010
Tomat and Lippard
Chart 1
Figure 1. Emission intensity changes (λex 463 nm) observed upon titra-
tion of RF3 (1.0 μM) with ZnCl2 (up to 600 equiv) in neutral aqueous
solution (50 mM PIPES buffer, 100 mM KCl, pH 7.0, 25 °C).
Scheme 1
Figure 2. Zinc response of RF3 as a function of pH in buffered aqueous
solutions (100 mM KCl, 25 °C, λex 463 nm). Formation of a white
precipitate was observed after the second zinc addition (final zinc
concentration 500 μM) at pH 8.5 and 9.0; intensity values under these
conditions were therefore not recorded.
Coordination of the divalent zinc ion would in turn repel the
positive charge and modulate the relative populations of the
iminophenoxide and aminoquinone mesomers to the bond-
ing description of the fluorophore (Chart 1). Such zinc-
induced rearrangement of charge distribution was envisioned
to elicit a change in absorption and emission wavelengths.
For the synthesis of rhodol RF3 (Scheme 1), a precursor
containing the zinc-binding unit was constructed through
reductive amination and subsequent alkylation under stan-
dard conditions (Supporting Information). Condensation of
compound 2 with 20-carboxy-5-chloro-2,4-dihydroxybenzo-
phenone afforded ethyl ester rhodol precursor 3 and, among
other less abundant side products, compound 4, an unusual
rhodamine carrying two metal-binding units directly installed
on the fluorophore. Compounds 3 and 4 were isolated and
separately hydrolyzed to give RF3 and RA1, respectively.
Under simulated physiological conditions (50 mM aqu-
eous PIPES buffer, 100 mM KCl, pH 7.0), rhodol RF3
exhibits visible absorption and emission maxima at 514 and
540 nm, respectively. Zinc coordination prompts a hypso-
chromic shift of the absorption band to 495 nm, with a
decrease of the extinction coefficient from (9.5 ( 0.2) ꢀ 104 to
(4.4 ( 0.1) ꢀ 104 M-1 cm-1 (Supporting Information). In the
emission profile (Figure 1), zinc binding is accompanied by a
change of emission properties from a quantum yield of 0.62(
0.01 at λmax 540 nm to a quantum yield of 0.52 ( 0.01 at λmax
523 nm. As such, rhodol RF3 behaves as a ratiometric zinc
sensor and its fluorescence response can be measured by the
ratio of emission intensities at 523 and 540 nm (F523/F540).
Zinc-induced changes in the emission profile of RF3 were
monitored over the course of a fluorometric titration experi-
ment (Figure 1), and Benesi-Hildebrand analysisreavealeda
dissociation constant (Kd) of 22 ( 2 μM (Supporting In-
formation). Zinc detection was not perturbed by biologically
abundant Na(I), Ca(II), and Mg(II) ions. Several divalent
transition metals, namely Mn(II), Fe(II), Co(II), Cd(II), and
Hg(II), caused little or no interference (Supporting In-
formation); however, Ni(II) and Cu(II) ions quenched the
fluorescence emission such that the intensity ratio F523/F540
could not be measured. Because these metal ions are not
present in micromolar concentrations in typical biological
specimens, they will not limit future applicability of RF3 in a
variety of settings. Furthermore, the emission profile and zinc
response of RF3 are stable over pH values ranging from 5.5
to 9.0 (Figure 2), and therefore this probe does not suffer
from a common weakness of fluorescein-based sensors.
The rhodamine fluorochrome has been employed in numer-
ous fluorometric and colorimetric indicators of metal ions.20,21
(20) Kim, H. N.; Lee, M. H.; Kim, H. J.; Kim, J. S.; Yoon, J. Chem. Soc.
Rev. 2008, 37, 1465–1472.

Communication
Inorganic Chemistry, Vol. 49, No. 20, 2010 9115
aniline-fused DPA binding units.4 The zinc-induced response
of RA1 is not affected by millimolar concentrations of Na(I),
Ca(II), and Mg(II); however, divalent transition-metal ca-
tions interact with the metal-binding units and quench
fluorescence emission in part (Mn(II), Fe(II), Co(II), Cd(II),
and Hg(II)) or in full (Ni(II) and Cu(II)), depending on their
relative affinities and electronic structure (Supporting In-
formation). Conversely, the emission intensity of RA1 is not
affected by changes of pH in the 5.5-9.0 range, and the zinc
turn-off response is only mildly reduced at pH values below
6.5 (Supporting Information).
In summary, considerations of charge distribution on the
rhodol scaffold with respect to metal coordination led to the
preparation of sensor RF3, which behaves as a ratiometric
probe for zinc ions and indicates a promising direction in
sensor design using rhodol platforms. Additionally, standard
xanthene-forming condensation procedures afforded the
symmetric rhodamine side product RA1, a turn-off inten-
sity-based indicator for several divalent transition-metal
cations. Both RF3 and RA1 bind zinc in the micromolar
concentration range and are not affected significantly by pH
changes in aqueous solution. When used in conjuction with
fluorescein-based turn-on zinc indicators, RA1 could provide
a useful control signal that is complementary both in color
and in zinc response. Owing to their moderate affinity for
zinc, RF3 and RA1 should prove valuable for the study of
zinc-rich cellular compartments in a variety of organs,
including the pancreas, the olfactory bulb, and the hippo-
campus.
Figure 3. Emission intensity changes (λex 490 nm) observed upon titra-
tion of RA1 (1.0 μM) with ZnCl2 (up to 300 equiv) in neutral aqueous
solution (50 mM PIPES buffer, 100 mM KCl, pH 7.0, 25 °C).
A common sensing mechanism features the analyte-induced
opening of a nonfluorescent colorless rhodamine spirolactam
to give a fluorescent species. In the present study, the isolated
side product RA1 provided the opportunity to investigate a
novel rhodamine-based sensor design.
Rhodamine RA1 displays intense absorption (λmax 535 nm)
and emission (λmax 561 nm) bands characterized by a molar
absorptivity of (4.5 ( 0.1) ꢀ 104 M-1 cm-1 and a quantum
efficiency of 0.70 ( 0.01. Addition of ZnCl2 to a neutral
aqueous solution of RA1 causes considerable loss of its visible-
light absorption (Supporting Information) and emission fea-
tures (Figure 3). This behavior is consistent with formation of
a nonaromatic spirolactone species, in which the zinc-binding
units do not share a positive charge and are available for
coordination. According to this proposed reactivity, the sensor
maximizes metal coordination capabilities at the expense of
xanthene conjugation.
Acknowledgment. This work was supported by the
National Institute of General Medical Sciences (Grant
GM065519). The content is solely the responsibility of the
authors and does not necessarily represent the official
views of the National Institute of General Medical
Sciences or the National Institutes of Health. We thank
Drs. Daniela Buccella and Michael D. Pluth for valuable
discussions.
Binding data relative to fluorescence titrations of RA1
with ZnCl2 were fit to a 1:2 zinc-binding model by nonlinear
least-squares methods. The resulting Kd values of 0.34 ( 0.05
and 2.2 ( 0.4 μM are consistent with the moderate
zinc-binding affinity of RF3 and, in general, with those of
Supporting Information Available: Synthetic procedures
and product characterization data, zinc-binding analysis
methods, selectivity, and pH dependence charts. This materi-
al is available free of charge via the Internet at http://pubs.
acs.org.
(21) Han, Z.-X.; Zhang, X.-B.; Li, Z.; Gong, Y.-J.; Wu, X.-Y.; Jin, Z.; He,
C.-M.; Jian, L.-X.; Zhang, J.; Shen, G.-L.; Yu, R.-Q. Anal. Chem. 2010, 82,
3108–3113.