Calcium Uncaging with Visible Light

Article abstract of DOI:10.1021/jacs.5b11606

We have designed a nitroaromatic photochemical protecting group that absorbs visible light in the violet-blue range. The chromophore is a dinitro derivative of bisstyrylthiophene (or BIST) that absorbs light very effectively (ε₄₄₀ = 66,000 M^-1 cm^-1 and two-photon cross section of 350 GM at 775 nm). We developed a "caged calcium" molecule by conjugation of BIST to a Ca²⁺ chelator that upon laser flash photolysis rapidly releases Ca²⁺ in <0.2 ms. Using the patch-clamp method the optical probe, loaded with Ca²⁺, was delivered into acutely isolated mouse cardiac myocytes, where either one- and two-photon uncaging of Ca²⁺ induced highly local or cell-wide physiological Ca²⁺ signaling events.

Full text of DOI:10.1021/jacs.5b11606

Article
pubs.acs.org/JACS
Calcium Uncaging with Visible Light
Hitesh K. Agarwal,†,∥ Radoslav Janicek,§,∥ San-Hui Chi,‡ Joseph W. Perry,‡ Ernst Niggli,*,§
and Graham C. R. Ellis-Davies*,†
†Department of Neuroscience, Mount Sinai School of Medicine, New York, New York 10029, United States
§Department of Physiology, University of Bern, Bern CH 3012, Switzerland
‡School of Chemistry and Biochemistry, Center for Organic Photonics and Electronics, Georgia Institute of Technology, Atlanta,
Georgia 30332, United States
S
* Supporting Information
ABSTRACT: We have designed a nitroaromatic photo-
chemical protecting group that absorbs visible light in the
violet-blue range. The chromophore is a dinitro derivative of
bisstyrylthiophene (or BIST) that absorbs light very effectively
(ε440 = 66,000 M−1 cm−1 and two-photon cross section of 350
GM at 775 nm). We developed a “caged calcium” molecule by
conjugation of BIST to a Ca2+ chelator that upon laser flash
photolysis rapidly releases Ca2+ in <0.2 ms. Using the patch-
clamp method the optical probe, loaded with Ca2+, was
delivered into acutely isolated mouse cardiac myocytes, where
either one- and two-photon uncaging of Ca2+ induced highly
local or cell-wide physiological Ca2+ signaling events.
these probes are very important additions to the arsenal of
caged compounds available for use by biologists, however none
of them offer the same generality of carbon−heteroatom bond
scission that is part of ortho-nitrobenzyl photochemistry.
Extended π-electron systems have been used to tune the
absorption maximum of fluorophores for one- and two-photon
(or 1P and 2P) excitation.25−29 However, when these
molecules are used for uncaging,25,27 they do not preserve
the generality of simple ortho-nitrobenzyl groups. Thus, we
have taken a step to address this need by creating an ortho-
nitrobenzyl caging chromophore with an absorption maximum
that is relatively bathochromic compared to the ortho-
nitroveratryl chromophore. The new chromophore, a dinitro
derivative of bisstyrylthiophene (or BIST, blue substructure of
1 in Figure 1), has a 1P absorption maximum at 440 nm
(Figure 2a) and is very sensitive to linear excitation across the
400−500 nm range of the electromagnetic spectrum. Further, it
has large 2P absorption cross section, of at least 250 GM, in the
720−830 nm range (Figure 2b). Here we detail the application
of the BIST chromophore to the development of a photo-
sensitive, Ca2+-selective chelator based on ethylene glycoltetra-
acetic acid (or EGTA),30,31 which, with the addition of the
cation, becomes a caged calcium probe (Figure 1) that is
activated very efficiently by visible light.
INTRODUCTION
■
Photochemical uncaging of organic substrates is a valuable
optical method that is used in many areas of science.1 Invented
by chemists for organic synthesis in the 1960s,2 it was adopted
by physiologists in the 1970s3 to control the concentration of
cellular signaling molecules.4 Material chemists have also found
an important application of this method to construct high-
density micro array chips.5 Uncaging remains a uniquely
powerful way to control the concentration of intracellular
calcium ions ([Ca2+]i), but since covalent bonds cannot be
made with ionized calcium, photolysis is normally used to
decrease in an irreversible manner the affinity of Ca2+ chelators
for the cation6,7 (note sodium8 and zinc9 uncaging use this
approach and that other approaches for Ca2+ have been
suggested recently).10,11 We have used the bifurcation of
known high-affinity tetracarboxylic molecules into known low
affinity dicarboxylates for highly efficient, rapid Ca2+ uncag-
ing.12−14 Because this is the only optical method for creating
large changes in [Ca2+]i of 10−100 μM, our optical probes have
been extensively used for many biochemical and physiological
experiments.15
The most widely used chromophores for all uncaging
experiments are the ortho-nitrobenzyl and ortho-nitroveratryl
protecting groups, which absorb light in the near-ultraviolet
range but are not efficiently photolyzed by visible light of longer
wavelengths. Subsequently several chromophores have been
used for uncaging of biological signaling molecules with blue
light.16−21 Photochemical protecting groups sensitive to green
light have also been reported,22−24 but to our knowledge no
biological studies have appeared with these probes. Some of
Received: November 5, 2015
© XXXX American Chemical Society
A
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Figure 1. Synthesis of BIST-2EGTA. Reagents and conditions: (a) Carboxymethyl-4-cyanophenylmethylsulfonium trifluoromethanesulfonate,
Cs2CO3, THF; (b) diethylene glycol, NaH; (c) CBr4, PPh3, DCM; (d) NaN3, NaI, DMF; (e) Pd(PPh3)4, 2,4,6-trivinyl-boroxin pyridine complex,
K2CO3, DME, H2O; (f) dibromothiophene, Pd(OAc)2, LiCl, TBACl, NaHCO3, DMF; (g) PPh3, dioxane, NaOH (aq); (h) BrCH2COOEt,
pentamethylpiperidine, acetonitrile; (i) KOH, MeOH (note for clarity the countercation is not shown); (j) CaCl2 in H2O (note for clarity the ionic
valence and counterion are not depicted).
Figure 2. Photochemical characterization of BIST-2EGTA. Nonlinear absorption properties of BIST derivatives were measured using the z-scan
technique or by 2P fluorescence emission. Rapid changes in [Ca2+]free were monitored in point scan mode (10 μs per pixel) or in bidirectional line
scanning mode (244 μs per line) using laser-scanning confocal microscopy at 561 or 473 nm after 2P photolysis with a mode-locked Ti:sapphire
laser tuned to 810 or 720 nm. (a) Absorption spectra of the BIST-2EGTA (blue) and ortho-nitroveratryl (DM-nitrophen, violet) chromophores
showing their relative 1P maxima. (b) 2P absorption spectra of BIST derivatives. Compound 9 had a 2P absorption maximum in DMSO of 740 GM
at 775 nm (orange). Each data point is an average of 5 or 6 measurements. Compounds 10a (R purple) and 10b (R green) have a 2P absorption
maximum in DMSO of 350 GM at 775 nm. All points are shown SD. (c) Example of Ca2+ titration of a solution of BIST-2EGTA with X-rhod-1.
Addition of 0.1 mM amount of CaCl2 to a solution of BIST-2EGTA (0.5 mM) at pH 7.2 with KCl (100 mM) showed the chelator had a high
prephotolysis affinity for Ca2+. (d) 2P uncaging of BIST-2EGTA produced a rapid increase in Ca2+ monitored by point scan confocal imaging using
rhod-FF. The exponential time-constant for the fluorescence increase was 164 μs. (e) The relative efficacy of 2P uncaging of DM-nitrophen at 810
and 720 nm was determined by monitoring the photoreleased Ca2+ during a power train at these two wavelengths. Both wavelengths showed a
quadratic dependence on incident power, and release was 7.4 0.23 times more effective at 720 nm (closed red diamonds) compared to 810 nm
(open black diamonds). Fluo-3 was used to monitor Ca2+ release. (f) Ca2+ release from BIST-2EGTA:Ca2+ complex at 810 and 720 nm was
determined by monitoring the fluorescence signal from rhod-FF during a power train at these two wavelengths (810, open black squares; 720 closed
red squares). The increase in fluorescence showed a quadratic dependence on incident power and was equally effective at both wavelengths. An
identical power train was also used for photolysis of DM-nitrophen (720, closed red diamonds). The resting [Ca2+]free, the Ca2+-bound, and Ca2+-free
indicator concentrations were the same for both caged calcium compounds.
synthesis of this molecule (Figure 1), called BIST-2EGTA,
started by conversion of 2-nitro-5-bromobenyzaldehyde into
the epoxide with carboxymethyl-4-cyanophenylmethylsulfo-
nium trifluoromethanesulfonate in the presence of cesium
RESULTS
■
Chemical Synthesis. Concerned about the lipophilic
nature of the BIST chromophore, we chose as our first target
a symmetrical molecule bearing two EGTA chelators. The
B
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carbonate to give 2 in a 74% yield. The diether backbone of the
Ca2+ coordination sphere was created using base-catalyzed ring
opening of the epoxide with ethylene glycol to give the desired
diol 3 in 80% yield, along with 5% of the other regiomer. The
mixture of diols was converted to their dibromides using Ph3P
and CBr4. From this mixture pure dibromide 4 was easily
isolated by flash chromatography and then treated with sodium
azide to give 5 in 74% overall yield for the two steps. The vinyl
unit was added to the chromophore by treatment of 5 with
2,4,6-trivinyl-boroxin pyridine complex and tetrakis-
(triphenylphosphine)palladium to give 6 in a yield of 73%.
The complete chromophore was constructed by Heck coupling
of 6 with dibromothiophene to give 7 in 71% yield. The ethyl
ester of BIST-2EGTA was made by reduction of the tetraazide
7 to its tetraamine in 70% yield, followed by alkylation with
ethyl bromoacetate to give octaester 8 in 31% yield. Finally, the
esters were hydrolyzed with an excess of KOH to give the
target chelator BIST-2EGTA (1). Importantly, we have found
such solutions to be highly stable. Routinely we store all caged
compounds at −40 °C, and solutions of BIST-2EGTA have
been found to be stable for more than one year, as judged by
HPLC (same single peak). Furthermore, such solutions are
equally efficacious when used for uncaging inside cells.
One- and Two-Photon Absorption Properties of BIST.
Irradiation of BIST-2EGTA with visible light revealed that the
new photosensitive chelator was photolyzed slightly more
slowly than DEAC450-Glu (quantum yield 0.39).32 Thus,
HPLC analysis showed that BIST-2EGTA photolyzed with a
quantum yield of photolysis of 0.23 (Figure 1, Supporting
Information, shows representative HPLC traces). The
absorption maximum of BIST was bathochromically shifted
when compared to the widely used ortho-nitroveratryl (DM-
nitrophen33) photochemical protecting group (Figure 2a) such
that BIST-2EGTA showed peak absorption at 440 nm with an
extinction coefficient of 66,000 M−1 cm−1. We used the z-scan
method34 and 2P-induced fluorescence to determine the 2P
absorption properties of simple BIST derivatives 9 and 10
(Figure 2b). For simplicity we chose to examine molecules
without benzylic heteroatom substitutions as such function-
alities would be photolyzed during such measurements. The
simplest dinitro-BIST (9) had a 2P absorption cross section in
DMSO of 740 GM at 775 nm. This chromophore absorbed 2
photons strongly across the 720−900 nm range (Figure 2b). A
BIST derivative having substituents ortho to both nitro groups
(10) showed similar, large 2P absorption properties, with a
maximum 2P cross section of 350 GM at 775 nm (Figure 2b).
Note, the effects of aqueous solvent on the 2P cross section in
other reports of similar compounds are modest.35 The 2-fold
reduction of the 2P cross section of 9 is probably caused by
steric clash between the ortho substituents and the nitro groups
twisting the latter out of planarity with the aromatic ring system
in compound 10.
physiological Mg2+ concentrations (i.e., 1 mM) had no effect on
these values. Importantly, Ca2+ was shown to be a photo-
product of the BIST-2EGTA:Ca2+ complex upon irradiation
with visible light. Photolysis of a solution of BIST-2EGTA 85%
saturated with Ca2+ (1 mM cage with 1.7 mM Ca2+ at pH 7.35)
with a blue laser (473 nm) increased the [Ca2+]free from 0.2 to
20 μM measured with a Ca2+-selective electrode. HPLC
analysis showed 50% of BIST-2EGTA remained in this
experiment (Figure 2, Supporting Information). The large
increase in free [Ca2+] is to be expected as iminodiacetic acids
are known to have Ca2+ affinity of about 1 mM in the
physiological pH range.36 We modeled our reaction with
“Patcher’s Power Tools” and found that the increase in free
[Ca2+] must result from a decrease in affinity of approximately
20,000-fold (i.e., from 50 nM to 1 mM). It should be noted that
the affinities of BIST-2EGTA before and after photolysis are, as
expected, the same as the UV-light sensitive Ca2+ cage NP-
EGTA,30,31 a chelator that is structurally identical in terms of
Ca2+ binding.
Characterization of Dynamic Calcium Uncaging. The
rate of substrate release is important for many applications of
caged compounds, especially those concerned with Ca2+
signaling.30 Low-affinity fluorescent Ca2+ dyes allow the
[Ca2+] to be measured with temporal fidelity, as the rate-
limiting step for equilibration of the dye:Ca2+ complex is
determined by the off-rate of the dye.37 Fluorescence imaging
with a confocal microscope in point scan mode revealed that
rhod-FF (Kd = 19 μM) showed a rapid change in signal
(exponential time constant of <200 μs) when BIST-2EGTA
was photolyzed using 2P excitation at 810 nm (Figure 2d).
The widely used15 nitroaromatic caged calcium compounds
(e.g., DM-nitrophen33 or NP-EGTA31) photolyze much more
effectively at relatively short wavelengths (e.g., 2P excitation at
720 nm, or 1P excitation in the UV−C range) when compared
to longer wavelengths (i.e., >800 nm for 2P excitation and
400−500 nm for 1P excitation). Thus, DM-nitrophen showed a
relative 2P-uncaging efficacy of 7.40 for 720 versus 810 nm
(Figure 2e). Consistent with the 2P absorption spectrum
(Figure 2b), we found that BIST-2EGTA was equally sensitive
to 2P photolysis at these two wavelengths (Figure 2f).
However, when DM-nitrophen was photolyzed under the
same resting [Ca2+]free the fluorescence signal from rhod-FF
upon Ca2+ release from BIST-2EGTA was about 13.7× larger
than after release from DM-nitrophen at 720 nm (Figure 2f).
Figure 2e,f also implies that BIST is about 100× more effective
at 810 nm. Taken together these data show that BIST-2EGTA
binds Ca2+ with high affinity, absorbs light strongly, and is
photolyzed efficiently to create changes in [Ca2+] that are
potentially useful for physiological studies. We tested such
effectiveness in acutely isolated cardiac myocytes.
Two-Photon Uncaging of Ca2+: Photocontrol of Local
Ca2+ Signaling. In cardiac myocyte cells a small amount of
Ca2+ enters the cytoplasm upon depolarization and initiates
Ca2+-induced Ca2+ release from the sarcoplasmic reticulum
(SR) store. Such release events can remain highly localized or
initiate “Ca2+ waves” that propagate through the cell.38 These
signals were recorded as confocal line scan (x,t) images, where
the vertical axis shows the spatial dimension, while the
horizontal axis represents time. Individual myocytes were
loaded with BIST-2EGTA (0.5 or 1 mM) and rhod-2 (0.1 mM)
or X-rhod-5F (0.1 mM) via a patch pipet. 2P excitation at 810
nm produced localized Ca2+ transients that were considerably
larger (Figure 3a) than those produced by BIST-2EGTA
Calcium Binding and Release. Calcium titration of BIST-
2EGTA in aqueous solution was used to determine the
apparent dissociation constant at various pH values. Figure 2c
shows the increase in fluorescence emission from X-rhod-1
during addition of defined aliquots of Ca2+ (0.1 mM) to a
solution of BIST-2EGTA. These data allow the free [Ca2+] to
be determined, as previously described,30 and showed that the
chelator bound Ca2+ with high affinity (Kd 84 nM at pH 7.2, 50
nM at pH 7.35, and 19 nM at pH 7.5). In independent
experiments we used Ca2+-selective electrodes to measure the
Kd, and these data gave identical values. The presence of
C
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Figure 3. Localized control of Ca2+-induced Ca2+ release in cardiac myocytes using 2P photolysis. Single cardiac myocytes were loaded via a patch
pipet with BIST-2EGTA and rhod-2. Changes in [Ca2+]free were monitored in line scan mode (2.116 ms per line) using laser-scanning confocal
microscopy at 561 nm after 2P uncaging at the center of the line with a mode-locked Ti:sapphire laser tuned to 810 nm. Line scan data are displayed
as 3D surface plots (time in x, space in y, and fluorescence as F/F0 on a pseudocolor scale in z) with the corresponding 2D plots (time in x and space
in y) below each 3D panel. Scale bars are 1 F/F0, 5 μm and 50 ms. (a) Point 2P irradiation with 5 ms pulse (red bar) triggered local Ca2+-induced
Ca2+ release from the SR. (b) Photolysis of BIST-2EGTA produced highly spatially confined Ca2+ release. The cell was treated with caffeine (20
mM) to unload the Ca2+ from the SR. (c) Increasing pulse duration to 20 ms initiated a Ca2+ “mini” wave, with discrete Ca2+ release events apparent
beyond the initial uncaging location. (d) Reducing pulse duration to 1 ms produced rapid, efficient, and highly localized Ca2+-induced Ca2+ release.
(e) Pure photolytic release of Ca2+ from BIST-2EGTA during irradiation for 1 ms (cell treated with caffeine as in b).
Figure 4. Visible light and 2P excitation of BIST-2EGTA:Ca2+ complex in cardiac myocytes initiates intracellular Ca2+ waves. Single cardiac myocytes
were loaded via a patch pipet with BIST-2EGTA and X-rhod-5F or rhod-2. Changes in [Ca2+]free were monitored in line scan (x,t) mode (2.116 ms
per line) using laser-scanning confocal microscopy at 561 nm after 2P uncaging at the center of the line or by whole cell frame scan (202 ms per
frame, 406 × 96 pixels) imaging after uncaging with visible light. A mode-locked Ti:sapphire laser tuned to 810 nm was used for 2P excitation (20
mW, 20 ms). A 405 nm continuous-wave laser was used for uncaging with visible light (10 mW, 100 ms). Line scan data are displayed as 3D surface
plots (time in x, space in y, and fluorescence as F/F0 on a pseudocolor scale in z) with the corresponding 2D plots (time in x and distance in y)
below. (a) Rapid line scan confocal imaging revealed 2P excitation (20 ms, red bar) could initiate Ca2+ signals that propagated extensively in both
directions away from the initial uncaging position. Scale bars for units of 1 F/F0, 5 μm and 50 ms. (b) Uncaging with visible light (orange circle)
initiated a Ca2+ wave that propagated throughout the cell. Top left is a transmitted light image of the cell with the pipet seen as a shadow.
Pseudocolor images represent raw fluorescence intensity data. Frame sequence is top left to bottom left, followed by top right to bottom right. The
nucleus and patch pipet can be seen as bright structures in the left portion of the cell. The time stamp is from the beginning of each frame.
photolysis in caffeine-treated cells (Figure 3b). Caffeine
completely unloads Ca2+ from the SR, so allows the pure
photolytic Ca2+ signal to be detected. The difference between
the two signals therefore reflects the biological Ca2+ release
from the SR. Uncaging for longer periods triggered intracellular
Ca2+ “mini-waves” that caused separate release events a small
distance from the uncaging site (Figure 3c). In contrast, we also
found that very brief irradiation of BIST-2EGTA for 1 ms could
initiate highly localized Ca2+-induced Ca2+ release from the SR
(Figure 3d,e). Previously we have found that with DM-
nitrophen any Ca2+ release from the SR always required
substantially longer flash durations (ca. 50 ms).13,14,39−41
Photochemically Initiated Intracellular Ca2+ Waves
Using Visible Light and 2P Excitation. We found that 2P
excitation of BIST-2EGTA could also initiate strong Ca2+-
induced Ca2+ release processes that extended considerable
distances from the uncaging point. In Figure 4a we show using
line scan fluorescence imaging that 2P excitation can produce
such Ca2+ waves that propagate rapidly in both directions. Note
that such line scan imaging (x,t) has the advantage of a higher
imaging rate compare to full frame (x,y,t), but conveys limited
spatial information about the entire cardiac cell. Thus, we
combined visible light uncaging with full frame imaging which
allowed us to produce striking Ca2+ waves that propagated
throughout the cell (Figure 4b). These signals are similar to
those reported in many physiological studies of cardiac
myocytes (reviewed in refs 38 and 42). It should be noted
there was negligible optical cross talk between the 810 nm 2P
uncaging and 561 nm confocal imaging lasers under these
conditions. Specifically, imaging at 561 nm was performed with
D
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a
Table 1. Photochemical Properties of Caged Ca2+ Probes
probe
nitr-558
ϕ
εmax (nm)
ε405 (nm)
ε473 (nm)
ϕεmax
2PCS (GM)
τ (μs)
0.012
0.18
0.23
1.0
5500 (350)
4300 (350)
975 (350)
165
129
0
66
774
0.01
0.01
<0.001
1.0
400
26
DM-nitrophen33
NP-EGTA31
azid-159
0
31
0
224
15
33,000 (342)
18,400 (330)
66,000 (440)
840
0
0
33,000
12,900
15,200
2000
50
NDBF-EGTA14
0.7
312
0.6
b
BIST-2EGTA
0.23
54,000
46,000
350
164
a
b
Symbols and notes: ϕ, quantum yield; ε, extinction coefficient; 2PCS, 2-photon cross section; τ, Ca2+ release time constant. Measured in DMSO
for 10, aqueous solvents could reduce this value by 20−40%.
energies 0.1% of those used for photolysis with 405 nm.
Further, irradiation with EGTA in place of BIST-2EGTA
(Figure 3, Supporting Information), or without prior chelator
loading with Ca2+, did not produce any Ca2+ waves.
Importantly, cells displayed normal excitation−contraction
coupling, as seen in the cellular Ca2+ transient preceding
photolytic Ca2+ release, implying that BIST-2EGTA is nontoxic
inside cells (not shown).
CONCLUSIONS
■
BIST is a new photochemical protecting group that absorbs
visible light efficiently. Beyond our initial application to Ca2+
uncaging, since BIST uses similar photochemistry to the widely
used ortho-nitrobenzyl and ortho-nitroveratryl chromophores,
we suggest BIST could be used to uncage the widest variety of
functionalities. Our current data show that BIST-2EGTA is an
exceptionally photosensitive caged Ca2+ probe (Table 1),
making superb use of both 1P and 2P excitation. Importantly,
BIST-2EGTA is photolyzed by blue light, and the wide
availability of light sources in this region could make Ca2+
uncaging experiments attractive to many more laboratories.
DISCUSSION
■
We have developed an extended π-electron nitrobenzyl caging
chromophore, which we call “BIST”, that is photolyzed
efficiently with visible light in the violet-blue region. Several
recently developed caging chromophores are photolyzed in this
range,1 however these cages are somewhat limited by the
photosolvolysis reactions they use.32 In contrast, the intra-
molecular photoredox reaction used by BIST can be used for
carbon-amine and carbon-ether photoscission43 (see Figure 4,
Supporting Information where we show that amines and
alcohols are uncaged cleanly from BIST). We took advantage of
this unique feature by using BIST to cleave a C−N tertiary
amine bond at the heart of the Ca2+-selective chelator EGTA to
develop a caged Ca2+ probe that is photolyzed with visible light
and thus fabricate a caged Ca2+ probe with distinctive optical
properties (Table 1).
METHODS
■
Chemical Synthesis. See Supporting Information for full methods
and analytic details.
Calcium Affinity. The Ca2+ affinity of BIST-2EGTA was measured
as previously described by us for NP-EGTA.31 Briefly, a homemade
Ca2+-selective electrode was made using ETH-129. To a solution of
BIST-2EGTA (0.5−1.0 mM) in HEPES (10 mM) and KCl (100 mM)
was added incremental amounts (0.1 mM) of CaCl2, and the change in
[Ca2+]free was measured. Scatchard analysis of such titrations revealed
that at pH 7.2 the Kd was 82 nM, 50 nM at pH 7.35, and 19 nM at pH
7.5. In separate experiments we also used changes in fluorescence from
X-rhod-1 to measure the [Ca2+]free. Values for the Kd using these
methods were identical. Using homemade electrodes we found that
the [Ca2+]free of a solution of BIST-2EGTA (1 mM) with 1.7 mM
CaCl2 increased from 0.2 to 20 μM when 50% of the caged compound
was photolyzed in a quartz cuvette with a 473 nm laser. Patcher’s
Power Tools (MPI for Biophysical Chemistry, Gottingen Web site)
allowed us to model this reaction to account for the change if [Ca2+]free
showing the affinity for Ca must be 1 mM.
Originally ultraviolet lasers were used to photolyze ortho-
nitrobenzyl caged compounds rapidly.44,45 Subsequently flash
lamps with a filtered output were used.46,47 However, blue
lasers are now much more widely available than either of these
light sources, being standard on all confocal microscopes and
used extensively for “optogenetics”.48 Therefore, the develop-
ment of a new caging chromophore that is sensitive and
efficiently photolyzed in this region of the electromagnetic
spectrum is a potentially useful addition to the optical toolkit
available to chemists and biologists. Having an extinction
coefficient of 66,000 M−1 cm−1 (cf. fluorescein49 that has an
extinction coefficient of 77,000 M−1 cm−1), it absorbs light very
efficiently in the blue region, a property which is unique
compared to other caged Ca2+ probes (Table 1). Furthermore,
2P uncaging is also considerably more efficient than the ortho-
nitroveratryl caging chromophore (Figure 2), suggesting that
direct attachment of a large 2P antenna to the caged substrate,
rather than relying on resonance transfer,50 can be very effective
for photorelease. Typically most extended π-electron systems
examined by material chemists for 2P absorption are electron
rich, with symmetrical diamino derivatives being very popular.51
It is interesting that our electron-deficient dinitro-BIST
derivatives have similar 2P absorption properties to comparable
diamino derivatives,52 suggesting that polarization per se is the
crucial property for chromophores in this context.
Quantum Yield of Photolysis. The quantum yield of photolysis
of BIST-2EGTA was measured as before.31,32 Briefly, solutions of
BIST-2EGTA and DEAC450-Glu, each with an optical density of 0.4
at 410 nm, were photolyzed with a 410 nm laser. The time-course of
photolysis was followed by HPLC analysis. Each time point was
analyzed thrice, and the photolysis of each compound was also
performed three times. The HPLC showed that the rate of photolysis
of BIST-2EGTA was 60% of DEAC450-Glu with a 410 nm laser,
implying a quantum yield of 0.23.
2P Absorption Cross Section. The 2P absorption cross section, δ
(in GM = 1 × 10−50 cm4 s molecules−1 photon−1), of the compounds
examined was measured in the 730−930 nm wavelength range. We
used the 2P-induced fluorescence (2PF) method53 using 5 ns, 10 Hz
pulses (compounds 10a,b) and the z-scan technique using 60 fs, 1-kHz
pulses (compound 9). In 2PF experiment, an optical parametric
oscillator laser (Spectra Physics, premiScan) pumped by a Q-switched
Nd:YAG laser (Spectra Physics, Quanta-Ray Pro-250) was used as the
excitation source. The measurements were conducted in a regime
where the fluorescence signal showed a quadratic dependence on the
intensity of the excitation beam. The fluorescence signal was collected
by a lens and transferred via a fiber to a spectrometer (HORIBA
Scientific, iHR320) with a CCD camera (HORIBA Scientific,
Synapse). Coumarin 485 (ref 54, Sigma-Aldrich), rhodamine B, and
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bisstyrylbenzene (compound 8 in ref 55), whose 2P properties have
been well characterized in methanol, were used as the references in
2PF measurements. The samples were prepared in DMSO (Sigma-
Aldrich, spectrophotometric grade) at a concentration of 3−8 × 10−4
M, and the optical path length of sample cuvettes for 2PF
measurements was 1 cm. For z-scan measurements,56 an optical
parametric amplified laser (Spectra Physics, TOPAS) pumped by a
mode-locked Ti:sapphire regenerative amplifier system (Spectra
Physics, Solstice) was used as the excitation source. The excitation
beam for z-scan is spatially filtered as a near Gaussian beam with M2 <
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.5b11606.
NMR spectra and HR-MS for all new compounds (PDF)
AUTHOR INFORMATION
■
Corresponding Authors
*graham.davies@mssm.edu
*niggli@pyl.unibe.ch
2
1.1 and waist ω(HW1/e ) ∼ 40 μm. The excitation irradiance ranged
from 50 to 150 GW/cm2. The samples were prepared in DMSO (ca. 2
mM), and the optical path length of sample cuvettes for z-scan
measurements was 1 mm.
Author Contributions
∥These authors contributed equally.
Laser Flash Photolysis in Droplets. A mode-locked Ti:sapphire
laser (Mira 9000F, Coherent, Santa Clara, CA, USA) pumped by 8 W
solid-state Verdi V-8 (Coherent) was used for 2P excitation of BIST-
2EGTA or DM-nitrophen. Wavelength of the laser was set to 720 or
810 nm with pulse duration of ∼120 fs. Power of the laser was
adjusted by neutral density and polarizing filters and was measured at
the objective focal plane by a power meter (PM200 with sensor
S170C, Thorlabs, Newton, New Jersey, USA). The laser beam was
guided to the SIM scanner of the confocal microscope (Fluoview
1000, Olympus, Volketswil, Switzerland) operating in single point
excitation mode simultaneously with the main scanner. The photolysis
period was 1 or 20 ms, and the duration was controlled by an
electronic shutter LS3 (Vincent Associates, Rochester, NY, USA) and
triggered by the confocal microscope. The main scanner of confocal
microscope was operating in line scan mode. To record changes in
Ca2+ concentration fluo-3, rhod-2 (both Biotium Inc., Hayward, CA,
USA), X-rhod-5F (Life Technologies), or rhod-FF (Teflabs, Austin,
TX, USA) were excited at 473 or 561 nm, respectively. Solutions used
for droplet experiments were composed of (mM): for Figure 2d: 1
BIST-2EGTA, 0.1 rhod-FF, 2 CaCl2, 100 KCl, 10 HEPES, pH = 7.80;
for Figure 2f: 1 BIST-2EGTA, 0.1 rhod-FF, 2 CaCl2, 100 KCl, 10
HEPES, pH = 8.0; for Figure 2e: 2 DM-nitrophen, 0.5 CaCl2, 0.1 fluo-
3, 1 GSH, 5 K2ATP, 10 HEPES, 20 TEA-Cl, 120 L-aspartic acid, 120
CsOH, pH = 7.20; for Figure 2f: 2 DM-nitrophen, 2 CaCl2, 0.1 rhod-
FF, 100 KCl, 10 HEPES, pH = 7.20. Recorded images were analyzed
in MATLAB (MathWorks, Inc., Natick, MA, USA) and Igor Pro
(WaveMetrics, Inc., Portland, OR, USA).
Laser Flash Photolysis in Cardiac Myocytes. Cardiac
ventricular myocytes were isolated from C57Bl/6 mice, as described
before.57 Myocytes were whole-cell patch-clamped at a resting
potential −80 mV. A train of 5−10 prepulses from −40 to 0 mV in
the presence of 100 nM isoproterenol was applied to load the
sarcoplasmic reticulum with Ca2+. A photolytic pulse with a duration of
1−100 ms was applied 1−3 s after last conditioning pulse to release
Ca2+ from BIST-2EGTA. For 2P photolysis we used a Ti:sapphire
laser with a wavelength of 810 nm, and for single photon photolysis we
used a UV diode laser with wavelength 405 nm. Both laser beams were
guided to the SIM scanner, and data acquisition was the same as
described above. Myocytes were placed in a recording chamber in
external bath solution containing (mM): 140 NaCl, 5 KCl, 1 CsCl, 1.8
CaCl2, 0.5 BaCl2, 10 HEPES, 10 glucose, pH = 7.40. Pipettes were
filled with internal solution containing (mM): for Figures 3a−e and
4a: 0.5 BIST-2EGTA, 0.8 CaCl2, 0.1 rhod-2, 1 GSH, 4 K2ATP, 5
MgCl2, 10 HEPES, 20 TEA-Cl, 120 L-aspartic acid, 120 CsOH, 8
NaCl, pH = 7.50; for Figure 4b: 1 BIST-2EGTA, 1.5 CaCl2, 0.1 X-
rhod-5F, 1 GSH, 5 K2ATP, 10 HEPES, 20 TEA-Cl, 120 L-aspartic acid,
120 CsOH, 8 NaCl, pH = 7.40. Images in Figures 3a−e and 4a were
normalized, filtered with Gaussian (kernel [5 5]) and Wiener filters
(kernel [10 10]), and smoothed by cubic spline (p = 0.5 in MATLAB
“caps” function). Experiments were performed at room temperature.
All recorded images were processed and analyzed in MATLAB,
imageJ, and Igor Pro.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by grants from the US NIH
(GM053395 and NS069720) to G.C.R.E.-D., the AFOSR
MURI (FA9550-10-10558) to J.W.P., and the Swiss National
Science Foundation (31-156375 and the Microscopy Imaging
Center, or “MIC”) to E.N. We would like to thank Drs. Simon
Langenegger and Robert Haner for their help.
̈
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