Photoactivatable Sensors for Detecting Mobile Zinc

Article abstract of DOI:10.1021/jacs.7b12766

Fluorescent sensors for mobile zinc are valuable for studying complex biological systems. Because these sensors typically bind zinc rapidly and tightly, there has been little temporal control over the activity of the probe after its application to a sample. The ability to control the activity of a zinc sensor in vivo during imaging experiments would greatly improve the time resolution of the measurement. Here, we describe photoactivatable zinc sensors that can be triggered with short pulses of UV light. These probes are prepared by functionalizing a zinc sensor with protecting groups that render the probe insensitive to metal ions. Photoinduced removal of the protecting groups restores the binding site, allowing for zinc-responsive changes in fluorescence that can be observed in live cells and tissues.

Full text of DOI:10.1021/jacs.7b12766

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Photoactivatable Sensors for Detecting Mobile Zinc
†
,§
†,§
Chanan D. Sessler, Nathan W. Vogler, Daniel Y. Zhang,†
†
‡
Jacob M. Goldberg,† Fang Wang,
William H. Loucks, Thanos Tzounopoulos, and Stephen J. Lippard*
‡
,†
†Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139,
United States
‡
Pittsburgh Hearing Research Center, Department of Otolaryngology, University of Pittsburgh, 3501 Fifth Avenue, Pittsburgh,
Pennsylvania 15261, United States
*
S Supporting Information
release, occur on short time scales. Nonetheless, there are
certain circumstances in which such a fast zinc response can be
ABSTRACT: Fluorescent sensors for mobile zinc are
valuable for studying complex biological systems. Because
these sensors typically bind zinc rapidly and tightly, there
has been little temporal control over the activity of the
probe after its application to a sample. The ability to
control the activity of a zinc sensor in vivo during imaging
experiments would greatly improve the time resolution of
the measurement. Here, we describe photoactivatable zinc
sensors that can be triggered with short pulses of UV light.
These probes are prepared by functionalizing a zinc sensor
with protecting groups that render the probe insensitive to
metal ions. Photoinduced removal of the protecting groups
restores the binding site, allowing for zinc-responsive
changes in fluorescence that can be observed in live cells
and tissues.
12
problematic. Consider the case of Zinpyr-1 (ZP1). In cells
with low cytosolic zinc concentrations, such as HeLa, ZP1
localizes to the Golgi apparatus, where it detects exogenously
added zinc. If cells have high concentrations of cytosolic zinc,
the probe can be saturated with the ion before it reaches the
Golgi. For cells that secrete zinc or samples that need to be
maintained in zinc-rich media, the sensor can be completely
saturated with extracellular zinc before it even crosses the cell
membrane. These features are not specific to ZP1 and limit the
utility of many small molecule metal ion sensors, particularly
those used in applications for which it is essential that the
sensor be delivered to a programed location without detecting
an analyte during transit.
To overcome these challenges, we devised a sensor that
would not respond to zinc until it is selectively activated in a
biological sample. In this manner, the sensor could be delivered
to any site of interest in a cell or tissue sample without
detecting zinc ions encountered in transit. Upon reaching the
desired target, the construct could be selectively and quickly
activated to reveal a fast and tight binding zinc probe. With
these goals in mind, we designed a series of protected zinc
sensors based on the ZP1 scaffold that met all requirements.
We chose photocleavable protecting groups, which are widely
used in biological experiments because they can be readily
removed with short pulses of light and provide excellent spatial
and temporal control over the release of caged molecules
inc is an essential element for human health. Throughout
Z
the body, zinc is tightly bound to proteins either as a
1
catalytic cofactor or structural element. In some tissues,
particularly those of the brain, pancreas, prostate, and
mammary gland, zinc exists in ion pools that participate in
2
signaling cascades and regulatory networks. This so-called
mobile zinc acts as a brake to attenuate glutamatergic
neurotransmission in certain areas of the brain engaged in
3
−5
sensory perception, especially in auditory processing,
and
6
also as a signaling agent in fertilization. Despite much research,
the exact functional role of mobile zinc in these pathways is not
completely understood. A critical barrier to understanding the
role of mobile zinc is a lack of suitable probes for studying these
13
including fluorescein. This strategy has proved effective for
14
copper sensing. A similar approach has been used for
preparing caged DNAzymes that sense metal ions, including
zinc and lead, but this method requires treating cells with a
DNAzyme and transfection reagents for several hours prior to
7
systems with high spatiotemporal resolution.
Many probes are described in the literature, some of which
have been used to great advantage to study the role of zinc in
15
8
imaging, which may not be suitable for all applications.
biological processes. Probes that fluoresce exclusively in the
As shown in Scheme 1, our strategy requires masking the
ZP1 xanthene-ring oxygen atoms involved in zinc binding by
functionalizing them with bulky o-nitrobenzyl groups. This
modification forces the fluorescein scaffold to adopt a
nonfluorescent lactone form and, as supported by theoretical
calculations, disrupts the zinc-binding site (see Supporting
Information). Upon irradiation of the protected molecule with
presence of zinc are among the most common and can be
categorized as those derived from small molecule and protein-
based fluorophores. These sensors employ diverse mechanisms
for detecting zinc in biological systems, ranging from changes in
photoinduced electron transfer or Fo
̈
rster resonance energy
transfer to complete structural rearrangements that occur upon
8
−11
zinc binding.
zinc rapidly.
In almost all cases, the sensors respond to
Generally, a fast zinc response is an advantageous property
Received: December 5, 2017
because many zinc-signaling events, such as synaptic zinc
©
XXXX American Chemical Society
A
DOI: 10.1021/jacs.7b12766
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
Scheme 1. Removal of Photocleavable Protecting Groups
from ZP1 to Give an Active Sensor
of zinc, irradiation of the sensor in the absence of zinc also
elicited a substantial increase in fluorescence, which could lead
to false positive results in biological experiments. Such concerns
prompted us to exclude sensors 2 and 4 in further studies. In
these and separate experiments, we observed that all sensors,
including ZP1, decompose during extended periods of
irradiation with UV light, particularly in the absence of zinc,
and therefore sensors that can be activated rapidly and
specifically are especially advantageous. Sensors 1 and 3 meet
these requirements.
We conducted a variety of experiments to find robust and
reproducible imaging methods. Because the protected sensors
are not fluorescent, calculated fluorescence turn-on values (F/
F0) were extremely sensitive to small variations in initial
fluorescence (F ), including those that arise from inadvertent
0
activation of the sensor in trace amounts by stray light. It is
therefore important to conduct imaging procedures in the dark.
In a typical experiment, HeLa cells were incubated with the
sensor to be tested in dye-free, serum-free medium (DMEM)
ultraviolet light, ZP1 is released and exhibits its typical zinc-
sensing behavior.
18
We prepared four ZP1 derivatives (1−4) by modifying a
with a small amount of Pluronic F-127, a nonionic surfactant.
1
6
published fluorescein diether synthesis because direct
After 15 min, the medium was removed and the cells were
washed thoroughly with phosphate-buffered saline (PBS).
Fresh dye-free, serum-free DMEM was added to the culture
dishes and the cells were imaged immediately. Zinc pyrithione
dialkylation of ZP1 did not give the desired regioisomer
(
Scheme S3). For each sensor, we measured the absorbance
and fluorescence in the presence and absence of excess zinc
before and after irradiation at 254 or 380 nm, and we followed
the course of each reaction by HPLC (Figures S5−S20). In
these studies, 1, 2, and 3 were photoactivatable as anticipated,
but 4 was not. Representative results are shown in Figure 1.
(
ZnPT, a zinc ionophore complex) was added to the cells on
the microscope stage and, after several minutes, additional
images of the same cells were acquired for comparison.
Representative images for sensor 3 are shown in Figure 2. The
sensor was essentially nonfluorescent in cells, even in the
presence of high concentrations of zinc. Irradiation for 15 s on
the microscope stage using a standard DAPI filter set was
sufficient to activate the sensors with an average integrated
fluorescence turn-on of 19 ± 1. Addition of TPA led to an
approximately 60% reduction in fluorescence, a result typically
Figure 1. Emission spectra of 3 μM 3 in 5 mM PIPES, 10 mM KCl in
3
0% CH CN in H O (v/v), pH 7.0 at 298 K; in the presence of 10
3 2
μM ZnCl ; after irradiation for 240 s with 254 nm (left) or 380 nm
2
(
right) light; and after addition of 20 μM tris(2-pyridylmethyl)amine
(
TPA).
The protected sensor 3 was not fluorescent in the absence of
zinc. Essentially no increase in fluorescence was observed from
the same solution when ZnCl was added. The fluorescence
2
then increased considerably upon UV-irradiation, consistent
with removal of the protecting groups and restoration of the
fluorophore. Finally, addition of the zinc chelator tris(2-
17
pyridylmethyl)amine (TPA) to the samples reduced the
fluorescence. HPLC analysis of the cuvette solutions confirmed
that ZP1 was generated after irradiation with UV light. Similar
results were obtained for sensors 1 and 2.
In contrast, irradiation of the sensors in the absence of zinc
induced only modest increases in fluorescence. Only trace
amounts of ZP1 were detected by HPLC analysis under these
conditions. As expected, the fluorescence intensity did not
Figure 2. Differential interference contrast (DIC) and fluorescence
microscopy images of (i) HeLa cells treated with 1 μM 3; (ii) after
treatment with 10 μM zinc pyrithione (ZnPT); (iii) after UV
irradiation for 15 s with a DAPI filter set; and (iv) after subsequent
increase significantly upon addition of ZnCl . Sensor 4 did not
2
incubation with 13 μM TPA. The average integrated fluorescence
turn-on is shown with standard error (n = 82). All p-values <10−
20
.
exhibit a satisfactory response in any of the tested conditions.
Although sensor 2 was rapidly photoactivated in the presence
Scale bar = 25 μm.
B
DOI: 10.1021/jacs.7b12766
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
Figure 3. Fluorescence of 3 in brain slices containing the dorsal cochlear nucleus (DCN) after photoactivation. (A) 4× image of the DCN slice
left), with overlaid 8 × 8 photostimulation grid (40 μm spacing). 20× image of the DCN (right) with the overlaid grid. The grid was
(
photostimulated 5 times with 1 ms pulses (355 nm, ∼5.5 mW). (B) Heat map of normalized fluorescent signals. ΔF/F is the fluorescence change
after photostimulation divided by the initial fluorescence. (C) Comparison of the average photoactivated fluorescence of sensor 3 in the molecular
layer versus the deep layer (regions outlined by yellow boxes in B, n = 6, p <0.0001, unpaired t test).
1
7
observed for ZP1 in cells. Sensor 1 gave qualitatively similar
results in analogous experiments (Figure S25).
anatomically well-separated, the DCN is ideal for testing
photoactivation of the sensor. Acute DCN slices incubated with
3 were irradiated with 355 nm laser light in a square grid
pattern spanning both the zinc-rich molecular layer and the
zinc-lacking deep layer. As shown in Figure 3, after 5 ms of
irradiation, only sites in the molecular layer exhibited significant
increases in fluorescence intensity after photostimulation, while
sites in the deep layer did not. Importantly, the fluorescence
response was restricted to loci of direct excitation; fluorescence
signals from tissue between or outside those regions were not
affected. In contrast, the diacetylated derivative of ZP1
Cells treated with 3 in the absence of any added zinc
exhibited an approximately 5-fold increase in fluorescence after
irradiation. Addition of ZnPT produced an additional 25%
increase in fluorescence intensity that could be reversed by
addition of TPA. Although similar results were obtained with
sensor 1, we observed that irradiation of 1 in the absence of
exogenous zinc induced a stronger fluorescence response in
cells, on average, than irradiation of 3 (Figures S23−S24).
Moreover, the higher sensitivity of 1 to UV light relative to that
of 3 may lead to inadvertent activation by stray light. For these
reasons, we restricted subsequent studies to 3.
We were especially interested in testing 3 in systems in which
typical small molecule sensors, such as ZP1, tend to have
difficulty, specifically (A) under conditions in which cells must
be maintained in zinc-rich medium or (B) when cells have high
cytosolic concentrations of mobile zinc. To mimic condition A,
4
fluoresces brightly in the entire molecular layer. This proof-
of-principle experiment shows that 3 can be selectively
activated in live tissue slices with at least micrometer spatial
resolution and millisecond temporal resolution.
In conclusion, we synthesized photoactivatable zinc sensors
based on the ZP1 scaffold that can be used to image zinc in
cells and tissue with high spatiotemporal resolution. Because
the properties of the ZP1 family can easily be tuned
synthetically, we expect that the strategy presented here can
be used to prepare photoactivatable sensors with nanomolar to
millimolar zinc-binding affinities. Furthermore, we envision this
method to be generally applicable to the protection of
fluorescein-based sensors for other analytes, including copper,
we spiked the culture medium with ZnCl before adding the
2
sensors. ZP1 does not show appreciable cell permeability under
these conditions and addition of ZnPT did not trigger an
increase in fluorescence (Figure S29). Under similar conditions,
3
appears to cross the cell membrane readily. Irradiation of the
cells after addition of ZnPT induced an approximately 50-fold
increase in fluorescence that could be reduced by treatment
with TPA (Figure S26). In the absence of ZnPT, the
fluorescence increased by only 7.5-fold upon irradiation (Figure
S28).
In separate experiments, HeLa cells were treated with ZnPT
and then washed before addition of any sensor to test condition
B. Unlike when ZnPT is added to cells pretreated with ZP1 and
induces a fluorescence response localized to the Golgi
apparatus, in these experiments bright fluorescence was
observed throughout the entire cell body (Figure S30).
Irradiation of cells treated with 3 under these conditions,
however, revealed distinct, characteristic patterns of localized
fluorescence (Figure S27). These results confirm that 3 is a
superior sensor for imaging cells that must be maintained in
zinc-rich environments.
To evaluate our ability to control the activation of the sensor
with high spatiotemporal resolution, we tested 3 in live brain
slices. For this work, we examined the mouse dorsal cochlear
nucleus (DCN), a region of the brain that integrates signals
from auditory nerve inputs in the vesicular (synaptic) zinc-
lacking deep layer with information arriving from other areas of
the brain in the synaptic zinc-rich molecular layer (Figure
22−25
iron, and nitric oxide.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.7b12766.
■
*
S
Experimental methods, computational details, and
characterization data (PDF)
AUTHOR INFORMATION
Corresponding Author
lippard@mit.edu
■
*
ORCID
Jacob M. Goldberg: 0000-0002-8004-3769
Fang Wang: 0000-0002-9192-6858
Chanan D. Sessler: 0000-0002-9577-2016
Nathan W. Vogler: 0000-0002-1526-3079
Daniel Y. Zhang: 0000-0002-7975-1653
William H. Loucks: 0000-0003-3779-6201
Stephen J. Lippard: 0000-0002-2693-4982
Author Contributions
3
,4,19−21
§
3).
Because the zinc-lacking and zinc-rich layers are
These authors contributed equally.
C
DOI: 10.1021/jacs.7b12766
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by funding from National Institutes
of Health grants R01-GM065519 (to S.J.L.) and R01-
DC007905 (to T.T.) and fellowships F32-GM109516 (to
J.M.G.) and F31-DC015924 (to N.W.V.). D.Y.Z and W.H.L.
thank the MIT Undergraduate Research Opportunities
Program for support. The Massachusetts Green High Perform-
ance Computing Center is acknowledged for providing
computational resources.
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DOI: 10.1021/jacs.7b12766
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX