Rapid, photoactivatable turn-on fluorescent probes based on an intramolecular photoclick reaction

Article abstract of DOI:10.1021/ja204758c

Photoactivatable fluorescent probes are invaluable tools for the study of biological processes with high resolution in space and time. Numerous strategies have been developed in generating photoactivatable fluorescent probes, most of which rely on the photo-"uncaging" and photoisomerization reactions. To broaden photoactivation modalities, here we report a new strategy in which the fluorophore is generated in situ through an intramolecular tetrazole-alkene cycloaddition reaction ("photoclick chemistry"). By conjugating a specific microtubule-binding taxoid core to the tetrazole/alkene prefluorophores, robust photoactivatable fluorescent probes were obtained with fast photoactivation (～1 min) and high fluorescence turn-on ratio (up to 112-fold) in acetonitrile/PBS (1:1). Highly efficient photoactivation of the taxoid-tetrazoles inside the mammalian cells was also observed under a confocal fluorescence microscope when the treated cells were exposed to either a metal halide lamp light passing through a 300/395 filter or a 405 nm laser beam. Furthermore, a spatially controlled fluorescent labeling of microtubules in live CHO cells was demonstrated with a long-wavelength photoactivatable taxoid-tetrazole probe. Because of its modular design and tunability of the photoactivation efficiency and photophysical properties, this intramolecular photoclick reaction based approach should provide a versatile platform for designing photoactivatable fluorescent probes for various biological processes.

Full text of DOI:10.1021/ja204758c

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pubs.acs.org/JACS
Rapid, Photoactivatable Turn-On Fluorescent Probes Based on an
Intramolecular Photoclick Reaction
Zhipeng Yu, Lok Yin Ho, and Qing Lin*
Department of Chemistry, State University of New York at Buffalo, Buffalo, New York 14260-3000, United States
S Supporting Information
b
example is the development of photoactivatable azido-DCDHF
ABSTRACT: Photoactivatable fluorescent probes are in-
valuable tools for the study of biological processes with high
resolution in space and time. Numerous strategies have
been developed in generating photoactivatable fluorescent
probes, most of which rely on the photo-“uncaging” and
photoisomerization reactions. To broaden photoactivation
modalities, here we report a new strategy in which the
fluorophore is generated in situ through an intramolecular
tetrazole-alkene cycloaddition reaction (“photoclick chemistry”).
By conjugating a specific microtubule-binding taxoid core to
the tetrazole/alkene prefluorophores, robust photoactivata-
ble fluorescent probes were obtained with fast photoactiva-
tion (∼1 min) and high fluorescence turn-on ratio (up to
112-fold) in acetonitrile/PBS (1:1). Highly efficient photo-
activation of the taxoidÀtetrazoles inside the mammalian
cells was also observed under a confocal fluorescence
microscope when the treated cells were exposed to either
a metal halide lamp light passing through a 300/395 filter or
a 405 nm laser beam. Furthermore, a spatially controlled
fluorescent labeling of microtubules in live CHO cells
was demonstrated with a long-wavelength photoactivatable
taxoidÀtetrazole probe. Because of its modular design
and tunability of the photoactivation efficiency and photo-
physical properties, this intramolecular photoclick reaction
based approach should provide a versatile platform for
designing photoactivatable fluorescent probes for various
biological processes.
fluorophores by Moerner et al. that exhibit very high turn-on
ratios in fluorescence (up to 1270-fold) and are suitable for
super-resolution imaging of targeted proteins in live cells.12
We recently reported a bioorthogonal chemistry strategy for
fluorescent labeling of proteins in live cells based on a photoinduced
tetrazole-alkene cycloaddition reaction (“photoclick chemistry”).13
Fluorescent pyrazoline cycloadducts were formed after the reac-
tions, ensuring that only alkene- or tetrazole-pretagged proteins
were labeled. Because the cycloaddition rate was modest (k2 values
up to 10 MÀ1 À1), relatively high concentrations of reagents
s
(∼100 μM) were typically required for labeling in the intracellular
environment. For faster fluorescent labeling at reduced reagent
concentrations, we reasoned that an intramolecular photoclick
reaction of a prefluorophore containing both the tetrazole and
the alkene functionalities would greatly increase the reaction rate,
resulting in rapid turn-on fluorescence for more efficient protein
visualization in vivo. To demonstrate this approach, we report
the design, synthesis, and photophysical characterization of a
class of ligand-directed, photoactivatable, turn-on fluorescent
probes for the spatially controlled imaging of microtubules in
live mammalian cells.
We decided to design turn-on fluorescent probes for imaging
microtubules because (i) microtubules exhibit dynamic insta-
bility and are responsible for spatial organization and rapid
remodeling of the cytoskeleton during cell division,14 and (ii)
specific microtubule-binding molecules such as paclitaxel15 and
its fluorescent derivatives16 have been used in the study of
microtubule motility in vivo. A series of taxoidÀtetrazoles 1À4
were thus synthesized (Schemes S1ÀS4 in Supporting In-
formation) in which 7-β-alanyltaxol core17 is conjugated to the
para-position of the C-phenyl ring of two water-soluble tetrazoles
bearing an o-allyloxy group at N-phenyl ring via a variable flexible
linker (Scheme 1). We expect that, upon photoirradiation,
tetrazoles would undergo rapid cycloreversion reaction to gen-
erate reactive nitrile imine dipoles which spontaneously react
with the prealigned allyl group to form the pyrazoline fluoro-
phores. The substituted amines or amides on the N-phenyl ring
and the various linkers at the C-phenyl ring serve to fine-tune the
reactivity of the tetrazoles as well as the photophysical properties
of the resulting pyrazoline fluorophores. To our satisfaction,
nearly quantitative conversions for 1À4 were observed based
on HPLC analysis of the reaction mixtures after photoirradia-
tion using a 302 nm hand-held UV lamp (UVM, 0.16 AMPS,
2.3 mW/cm2)18 for 60À70 s (Figures S1ÀS4).
hotoactivatable small-molecule fluorescent probes have be-
P
come an indispensable tool for microscopic studies of
biomolecular dynamics in living cells, tissues, and animals.1
Several strategies have been reported for the design of photo-
activatable organic fluorophores, including reversibly photoacti-
vatable fluorophores based on a light-induced bond-breaking and
thermal bond-reforming process;2 photo-uncaging of “caged”
fluorophores such as fluorescein,3 coumarin,4 rhodamine,5
and FRET-based dyes;6 photorelease of fluorophores from
quenchers via cleavage of the photolabile linkers;7 and photo-
switching of the spiropyran-merocyanine-based nanoparticles.8
In these systems, the latent fluorophores are already in place,
which may compromise the photoactivation efficiency due
to the filtering effect.9 Also, recent advance in the super-resolu-
tion microscopic techniques such as PALM10 and STORM11 also
demands new strategies for designing photoactivatable fluoro-
phores with increased biocompatibility, excellent turn-on effi-
ciency, and greater flexibility in bioconjugation. An elegant
Received: May 24, 2011
Published: July 07, 2011
r
2011 American Chemical Society
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Scheme 1. Design of Photoactivatable Turn-On Fluorescent
Probes 1À4 for Microtubules Comprising of a 7-β-Alanyltaxol
Core and a Tetrazole Prefluorophore Core
Figure 1. Confocal spectrum-scan micrographs of taxoidÀpyrazoline
labeled microtubules in live CHO cells. CHO cells were treated with
1 μM oftaxoidÀpyrazoline1a(a andb) or3a(d ande) for30min and the
fluorescence spectrum-scan in the region of 428À608 nm (a and d) and
the corresponding DIC images (b and e) were recorded with a Zeiss LSM
710 microscope; λex = 405 nm. The colors rendered in the fluorescence
micrographs were λ-encoded (real color). Scale bar = 20 μm. The average
intensities of the unsaturated fluorescence-labeled cytoskeleton areas in
10 selected cells were plotted to give the in-cell fluorescence spectrum of
1a (c) or 3c (f) along with the standard deviations.
Table 1. Photophysical Properties of the TaxoidÀ
Pyrazolines and Fluorescence Turn-On Efficiencya
from 0.0024 and 0.0041 in ACN-mixed PBS buffer to 0.036 and
0.022 in DCM, respectively (compare Figure S6 to S5; Figure S8
to S7). Interestingly, taxoidÀpyrazolines 3a and 4a exhibited
significantly higher fluorescence quantum yields in ACN/PBS
buffer (1:1), with ΦF values to be 0.034 and 0.056, respectively.
Because of the difference in their brightness, the four taxoidÀ
tetrazoles showed varying degrees of fluorescence turn-on effect,
ranging from 21-fold for photoactivation of 1 to 112-fold for
photoactivation of 4 in ACN/PBS buffer (1:1) (Table 1).
It is known that the modification of paclitaxel on position-7
does not affect its binding to β-tubulins,16 the building blocks for
polymeric microtubules. To investigate whether this is also true
for our pyrazoline-modified taxoids, HeLa cells were treated with
5 μM of preactivated taxoidÀpyrazoline 1a for 30 min. After cell
fixation and permeabilization, the binding of taxoidÀpyrazoline
1a to microtubules was confirmed by confocal fluorescence
microscopy in which the distribution of pyrazoline fluorescence
overlaid nicely with that of an Alexa Fluor-568 based immunos-
tain for microtubules (Figure S11). Conversely, since the binding
of taxoid to β-tubulins may bring the hydrophobic pyrazoline
fluorophores to the microtubule surface, it is also possible that
the photophysical properties of taxoidÀpyrazolines may be
altered. To probe this possibility, CHO cells were treated with
1 μM of the preactivated taxoid-pyrazolines 1aÀ4a for 30 min,
and the taxoidÀpyrazoline fluorescence upon binding to micro-
tubules was examined with a spectrum-scan confocal microscope.
To our surprise, among the four taxoidÀpyrazolines, compounds
1a and 3a showed moderate and strong fluorescent labeling of
microtubules, respectively (Figure 1), while compounds 2a and
4a showed very weak intracellular fluorescence (Figure S12).
This drastic difference in fluorescent properties apparently is
dependent on the linker length; the long flexible linkers such as
those in 2a and 4a (Scheme 1) possibly position the pyrazoline
fluorophores in a protein environment where their fluorescences
are quenched. A closer examination of in-cell fluorescence
spectra revealed that the peak emission wavelengths have shifted
from 617 to around 569 nm for 1a (Figure 1c), and from 584 to
543 nm for 3a (Figure 1f). These hypsochromic shifts are
presumably due to the interactions of the pyrazoline fluoro-
phores with the microtubule surface, and have been observed
previously for the fluorescein-conjugated taxols.16b As expected,
λmax
ε
ε405
λem
fluorescence
compound (nm)b (MÀ1 cmÀ1)c (MÀ1 cmÀ1
)
(nm) ΦF
turn-onf
d
e
1
310
330
340
311
332
344
262h
358
276h
336
6400
5100
460
ND ND
-
1a
1ag
2
2000
1600
540
617 0.0024 21-fold
4400
580 0.036
ND ND
34-fold
-
12000
8700
2a
2ag
3
3000
4700
ND
614 0.0041 30-fold
11600
24400
12900
30300
16400
587 0.022
ND ND
584 0.034
ND ND
528 0.056
101-fold
-
3a
4
6900
ND
111-fold
-
4a
3100
112-fold
a Compounds were dissolved in ACN/PBS (1:1) to derive concentra-
b
tions of 25 μM unless noted otherwise. λmax in the 300À500 nm
region. c Extinction coefficient at λmax. d Extinction coefficient at 405 nm.
e Quantum yields were measured using DAPI as a standard,20 and
f
integrated over 415À790 nm. Obtained by comparing the emission
intensity of taxoidÀpyrazoline to that of the corresponding taxoidÀ
h
tetrazole at λem; λex = 405 nm. g Dissolved in DCM. λmax in the
250À500 nm region. ND = not detected.
To determine the magnitude of fluorescence turn-on effect,
the corresponding taxoidÀpyrazolines 1aÀ4a were purified to
homogeneity and their photophysical properties were measured
along with those of taxoidÀtetrazoles 1À4 (Figures S5ÀS10).
As shown in Table 1, the N, N-dihydroxyethylamino substituted
taxoidÀtetrazoles (1 and 2) showed a bathochromic shift in λmax
compared to the glycolamide substituted ones (3 and 4) in
acetonitrile (ACN)/PBS buffer (1:1), consistent with what we
observed previously.19 However, taxoidÀtetrazoles 3 and 4
showed higher coefficients at their λmax. On the other hand,
the taxoidÀpyrazolines showed significant bathochromic shift,
with λmax ranging from 330 to 358 nm. Whereas none of the
taxoidÀtetrazoles was fluorescent, broad emission spectra were
obtained for all taxoidÀpyrazolines, with λem ranging from
528 nm for 4a to 617 nm for 1a. The fluorescent properties of
taxoidÀpyrazolines appeared to be solvent-dependent; for ex-
ample, the fluorescence quantum yields of 1a and 2a jumped
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Figure 2. Timecourses ofphotoactivationoftaxoidÀtetrazoles 1 (a) and
3 (b) as monitored by UVÀvis (dashed lines) and fluorescence (solid
lines). Compounds were dissolved in acetonitrile/PBS (1:1) to derive
concentrationsof25μM, andthe photoirradiation wavelengthswereset at
365 nm for 1 and 302 nm for 3. For fluorescence, λex = 405 nm.
Figure 3. Time course of the photoactivation of taxoidÀtetrazole 3 in
live CHO cells. (a) Time-lapsed confocal micrographs of tetrazole-treated
CHO cells after photoillumination, scale bar = 20 μm. Cells were treated
with 1 μM of taxoidÀtetrazole 3 for 30 min before photoillumination.
ZEISS filter set #49 was used for incident light filtering; for fluorescence,
λex = 405 nm. (b) Plotofthe changes of fluorescenceof3aovera period of
30 s. For each time point, the fluorescence intensities inside the cytosols of
10 individual cells with unsaturated fluorescence were used in the
calculation of the averaged intensities and the standard deviations.
taxoidÀpyrazoline 3a is a brighter fluorescent probe than 1a as it
showed a higher fluorescence intensity inside CHO cells.
On the basis of the in-cell spectrum scan results, taxoidÀ
tetrazoles 1 and 3 were selected and their photoactivation
kinetics in vitro were examined (Figure 2). Because taxoidÀ
tetrazole 1 showed broad absorption, we carried out the photo-
activation using a 365 nm hand-held UV lamp (3.5 mW/cm2).
Figure 2a shows that a complete conversion to the desired
taxoidÀpyrazoline 1a was achieved in 320 s, as evidenced by
the gradual increase in a new absorption band centered at 330 nm
and a broad emission band centered at 617 nm. On the other
hand, since taxoidÀtetrazole 3 showed almost no absorption at
365 nm, it was subjected to 302 nm photoirradiation (2.3 mW/
cm2). Gratifyingly, complete conversion to fluorescent taxoidÀ
pyrazoline 3a was achieved in 40 s, with the pyrazoline product
showing an absorption band at 358 nm and a strong emission
band at 584 nm (Figure 2b). Overall, these two taxoidÀ
tetrazoles offer complementary features as photoactivatable
turn-on fluorophores: tetrazole 3 exhibits robust turn-on effect
and higher brightness, while tetrazole 1 can be activated with
long-wavelength light (365 nm), which makes it potentially
suitable for laser-scanning fluorescence microscopy studies with
improved spatiotemporal resolution.
To examine whether taxoidÀtetrazoles can be photoactivated
in vivo, CHO cells were treated with 1 μM of taxoidÀtetrazole 3
for 30 min, and after washing with prewarmed PBS three times,
the treated cells were illuminated continuously with a metal
halide light source (EXFO-XCite, 25% of 13.3 mW/cm2)
through a filter with bandwidth of 300/395 nm, and the in situ
formed taxoidÀpyrazoline fluorescent signals were recorded
with an acquisition window of 535À545 nm. A rapid, roughly
12-fold increase in fluorescence in the CHO cytosols was
detected after 5-s photoirradiation (compare fluorescence in-
tensity at 5 s to background fluorescence at 0 s; Figure 3). By
comparing the fluorescence intensity at 5 s to the maximal
intensity for the taxoidÀpyrazoline 3a (Figure 1f), a calculated
79% yield was derived for the photoactivation in vivo. The
fluorescence intensities decreased over extended photoirradia-
tion; however, 56% of fluorescence signals still remained after 30
s photoirradiation, indicating that the pyrazoline fluorophore 3a
is fairly resistant to photobleaching. The photobleaching rate of
3a was similar to that of DAPI measured under identical
microscopic conditions (Figure S13).
Since taxoidÀtetrazole 1 shows 365-nm photoactivatability
and its UVÀvis absorption extends to 405 nm (albeit weak), a
laser wavelength available in our microscope, we decided to
examine whether photoactivation can be carried out with the
405-nm laser, which would allow a spatiotemporal control over
the in situ fluorescence generation. Thus, CHO cells were treated
with 10 μM taxoidÀtetrazole 1 for 30 min and then washed with
prewarmed PBS three times. Then, a select subpopulation of cells
in the optical field (area outlined in red rectangle in Figure 4)
was subjected to a brief (11 s) 405-nm diode laser irradiation
(30 mW) at 100% power while the cells outside the selected area
were not. Afterward, the entire population was scanned for
fluorescence emissions with excitation at 405 nm at 20% power.
Strong turn-on fluorescence was detected for cells within the rect-
angular area, in sharp contrastto theweakfluorescence background
for cells outside the activation area (Figure 4). Quantification of
cytosolic fluorescence intensity in CHO cells revealed a 7-fold
enhancement in fluorescence for the activated cells relative to the
unactivated cells. With this photoactivatable microtubule fluores-
cent probe, in principle it should be possible in the future to label
microtubules asymmetrically within a single cell and identify factors
that break cellular symmetry during the cell division.21
In conclusion, we have demonstrated a new strategy for
designing turn-on fluorescent probes in which a protein-targeting
core is linked to a photoactivatable, alkene-appended tetrazole.
Using the tetrazole-modified taxoids as a model, robust fluores-
cence turn-on (up to 112-fold) was observed within ∼1 min after
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National Science Foundation Major Research Instrumentation
grant (DBI-0923133) for instrumentation support.
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’ AUTHOR INFORMATION
Corresponding Author
qinglin@buffalo.edu
’ ACKNOWLEDGMENT
We gratefully acknowledge the NIH (R01 GM 085092) for
financial support, Alan Siegel at the SUNY Buffalo Biological
Sciences Imaging Facility for assistance in microscopy, and the
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dx.doi.org/10.1021/ja204758c |J. Am. Chem. Soc. 2011, 133, 11912–11915