New photolabile BAPTA-based Ca2+ cages with improved photorelease

Article abstract of DOI:10.1021/ja2115184

The efficient synthesis, physicochemical and photolytical properties of a photoactivable BAPTA-based Ca²⁺ cage containing two photosensitive o-nitrobenzhydryl groups attached to the aromatic core are described. Ca ²⁺ release in livin

Full text of DOI:10.1021/ja2115184

Article
pubs.acs.org/JACS
New Photolabile BAPTA-Based Ca2+ Cages with Improved
Photorelease
Jiaxi Cui,† Radu A. Gropeanu,† David R. Stevens,‡ Jens Rettig,‡ and Aranzazu del Campo*,†
́
†Max-Planck-Institut fur Polymerforschung, Ackermannweg 10, 55128 Mainz, Germany
̈
‡Physiologisches Institut, Universitat des Saarlandes, 66421 Homburg, Germany
̈
S
* Supporting Information
ABSTRACT: The efficient synthesis, physicochemical and photolytical properties of
a photoactivable BAPTA-based Ca2+ cage containing two photosensitive o-
nitrobenzhydryl groups attached to the aromatic core are described. Ca2+ release
in living cells was evaluated. The double substitution with the chromophores caused
a significant improvement of the Ca2+ release properties of nitr-T versus singly
substituted reported nitr-x derivatives without compromising Ca2+/Mg2+ selectivity
or pH insensitivity. Our results demonstrate a general strategy to improve light-
triggered Ca2+ release which may result in more efficient, selective, and pH-
insensitive photolabile Ca2+ chelators.
for calcium. This fact limits the applicability of these
compounds in some cases.
INTRODUCTION
■
Ionic calcium (Ca2+) is one of the most important second
messengers in cells.1 Localized fluctuations of intracellular free
Ca2+ concentration can regulate a variety of cellular functions,
e.g., nerve impulses, muscle contraction, secretion, ion channels
in the plasma membrane, or nonmuscle motility. To obtain
sudden jumps of intracellular calcium ions, different photolabile
caged Ca2+ molecules have been developed during the past 25
years.1−3 Light irradiation of calcium cages releases free Ca2+ in
solution and triggers the biological response. Caged Ca2+
compounds are now a well established working tool for studies
in cellular physiology and neurobiology.1
Photolabile BAPTA-based chelators (nitr-x) do not show
pH-sensitivity in the physiological range, have high Ca2+/Mg2+
selectivity (∼105), but they release Ca2+ rather ineffi-
ciently.11−13 The light-induced affinity changes of nitr-x
compounds is enhanced by the electron withdrawing properties
of the substituents on the aromatic ring of the photoproduct.
Reported nitr-x variants include one photosensitive group
attached to one of the two BAPTA rings, though the possible
benefit of including a second photosensitive substituent for
improving the effectiveness of Ca2+ release has been
indicated.14 Following the double substitution concept, we
describe the efficient synthesis and improved properties of
BAPTA-based cages containing two photosensitive o-nitro-
benzhydryl groups (nitr-T, Scheme 1) and evaluates their Ca2+
release in living cells. We demonstrate a general strategy to
improve light-triggered Ca2+ release in BAPTA-chelators
without compromising other properties. This strategy may
open a door to preparation of more efficient, selective, pH-
insensitive, and photolabile Ca2+ chelators.
When designing photolabile Ca2+ chelators, different require-
ments need to be considered: (1) chemical yield of Ca2+
release; (2) cation binding selectivity; (3) effectiveness of light
absorption (the extinction coefficient and 2-photon cross
section) and photochemical yield; (4) rate of Ca2+ release and
(5) pH sensitivity of cation binding. So far, available photolabile
Ca2+ chelators are not able to satisfy all of these requirements.1
EDTA-based chelators like DM-nitrophen delivers Ca2+ very
efficiently upon photolysis, but has a low Ca2+/Mg2+ selectivity
(500-fold) and this limits its application for intracellular
delivery.4,5 EGTA-based chelators feature high Ca2+/Mg2+
selectivity (∼105),6 and their efficiency has been remarkably
improved over the last 20 years7−10 to finally obtain a NDBF-
EGTA cage with excellent performance.7 These EGTA-based
chelators (NP-EGTA, NDBF-EGTA) allow fast Ca2+ release
(see Table S1 in Supporting Information, SI, for kinetic data),
which makes them currently the best choice in most
experiments in neurobiology research.1 However, the inherent
sensitivity of the EGTA binding moiety to pH changes in
physiological range (6.5−7.5) may result in a slower or
incomplete recovery to resting Ca2+ after illumination since the
acidification caused by uncaging changes the affinity of the cage
RESULTS AND DISCUSSION
■
Synthesis. The chemical structure of nitr-T is shown in
Scheme 1. Two o-nitrobenzhydryl groups were attached to the
p-positions of BAPTA. For comparison the single substituted
derivative (nitr-S) was also prepared. The nitr-T or −S
tetraethyl esters were obtained by trimethylsilyl trifluorome-
thanesulfonate assisted coupling of BAPTA tetraethyl ester to
5-methoxy-2-nitrobenzaldehyde followed by treatment with
tetrabutylammonium floride trihydrate (Scheme 1).12 The
intermediate esters were stable in solid state (no photolysis
Received: December 9, 2011
Published: April 22, 2012
© 2012 American Chemical Society
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nitr-T at the same wavelengths. This agrees with the fact that
nitr-S molecule contains a single o-nitrobenzhydryl group.
The photolysis reaction of nitr-T changed the UV spectrum
and a new maximum at 380 nm appeared (Figure 1), in
agreement with reported UV spectra of photolyzed nitr-5.12
The new peak was attributed to the formation of 4-amino-2′-
nitrosobenzophenone photoproducts (Scheme 2).12 It is
Scheme 1. Synthesis of Nitr-T and Nitr-S
Scheme 2. Photolytic Reactions of Nitr-T and Its Calcium
Complex Illustrating the Photolysis Intermediates
after storage at room temperature for 14 days), but they
photolyzed after a few hours when they were stored in solution
under laboratory conditions (a CDCl3 solution at room
temperature showed 25% photolysis after 4 h). For this reason,
immediate purification after reaction is necessary in order to
obtain high yields after isolation. Hydrolysis of the ethyl esters
in aqueous NaOH gave nitr-T and nitr-S. By using this
synthetic route only two steps are required to obtain the nitr-
T/S derivatives in good yields (total yield is 88% for nitr-T and
48% for nitr-S) and in a high purity of the crude products (e.g.,
nitr-T: >95%). In comparison, the reported nitr-5 required six
synthesis steps with a total yield about 12%,12 while NP-EGTA
required 10 steps and the total yield was 24%.8 Our synthesis of
nitr-T/S derivatives was much simpler and efficient.
important to note that the photocleavage of nitr-T involves
first the formation of a nonsymmetric (mononitrosobenzophe-
none)−(mononitrobenzhydryl) intermediate, which further
photolyses to give the final photoproduct. To confirm this
mechanism, the intermediate and final product were isolated
from photolyzed mixtures and validated (see Experimental
Section and Figures S10 and S11 of the SI). Therefore, three
different species are present during exposure (Scheme 2).
When the photolysis was performed in the presence of Ca2+, a
weaker maximum at 380 nm was detected which was attributed
to the electron-withdrawing effect of the coordinated Ca2+ on
the amine group of the chromophore.15
Photochemical Properties. Figure 1 presents the UV−vis
spectra of nitr-T under different conditions. The absorbance
The photolytic conversion of nitr-T in the absence and
presence of Ca2+ with increasing exposure was followed by
UV−vis spectroscopy (Figures S2 and S3 of the SI and Figure
2a) and the composition of the photolyzed mixture for the
different conversions was monitored by 1H NMR spectroscopy
(Figure S4 of the SI). The photolysis of nitr-T was quantified
by the disappearance of the methylidyne proton (a) at 6.28
ppm. The ethylene protons (b) shift from 4.19 ppm (b1) in
Figure 1. UV−vis spectra of a 9 μM solutions (40 mM Hepes/100
mM KCI, pH 7.2) of nitr-T, nitr-T with 240 mM CaCl2, isolated
photolyzed intermediate of nitr-T, photolyzed intermediate of nitr-T
with 240 mM CaCl2, isolated final photolyzed nitr-T photolyzed and
final photolyzed nitr-T with 240 mM CaCl2. See Scheme 2 for the
chemical structures.
coefficients of nitr-T at 267, 350, and 360 nm in Ca2+-free
buffer (40 mM Hepes/100 mM KCI, pH 7.2) were 2.75 × 104,
8.23 × 103, and 6.15 × 103 M−1cm−1, respectively. Upon
addition of Ca2+ (excess), the Ca2+ complex was formed and
this was reflected by a significant decrease of the absorbance at
267 nm and an increase at 350 nm. Nitr-S presented a similar
spectrum (see Figure S1 of the SI). The extinction coefficients
of nitr-S were 4.51 × 103 and 3.4 × 103 M−1cm−1 at 350 and
360 nm, respectively, which represent nearly half the values of
Figure 2. (A) Photolysis conversion (100%) versus irradiation doses
of nitr-T and nitr-T-Ca2+ complex (9 μM of nitr-T and 18 μM of nitr-
T-Ca2+ complex in 40 mM Hepes/100 mM KCI with pH 7.2). Ten
mM Ca2+ was added for nitr-T-Ca complex and a monochromatic light
of 360 nm (2.77 × 10−9 E/s) was used. (B) Composition of a nitr-T
solution during photolysis. The amount of the reagent and the
photoproducts was determined from the relative height of the ethylene
1H NMR signals at 4.19, 4.24, and 4.28 ppm (b1, b2, and b3 in Figure
S4 of the SI). The conversion was calculated from the integral of the
proton signals at 7.04 and 7.28 (c1 and c2 in Figure S4 of the SI).
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nitr-T to 4.24 ppm (b2) in the intermediate product and to
4.28 ppm (b3) in the final photoproduct. The aromatic proton
(c) shifted from 7.06 ppm (c1) to 7.27 ppm (c2) upon
irradiation and this was attributed to the appearance of the
conjugated structure. Since no other signals were observed in
the NMR spectra, it was assumed that nitr-T converted to the
final photoproduct nearly quantitatively. In fact, a chemical
yield for the photoreaction of 94% was determined by HPLC.
The composition of the photolyzed mixture at a given
1
conversion was calculated from the H NMR signals integrals
(Figure 2). During the exposure, the concentration of nitr-T
decreased while the concentration of the final photoproduct
increased, and the concentration of the photolysis intermediate
increased first and decreased after about 50% conversion.
The quantum yield of the photolytic reaction was calculated
by ferrioxalate actinometry. A quantum yield (Φ360) of 0.056
was obtained for the nitrobenzhydryl group in nitr-T. In the
presence of Ca2+ ions, the determined Φ360 value was doubled
(0.12). Nitr-S and nitr-S-Ca2+ complex showed a similar
tendency of their quantum yields (0.06 for nitr-S and 0.11 for
its calcium complex). It is important to note that these values
are considerably higher than the reported Φ360 data of nitr-5
and nitr-7 (0.012 and 0.011, respectively).1 We attribute this
difference to the different methods used for quantum yield
determination and the inherent error in quantum yield
measurements.16 The Ca2+-induced improvement in quantum
yield was also observed in nitr-5,12 but not in EGTA-based
chelators (Φ350 of NP-EGTA and NP-EGTA-Ca2+ complex
were 0.23 and 0.20, respectively).8 The photolysis reaction of
nitrobenzyl compounds involves the formation of a key aci-
nitro intermediate.17,18 In the case of nitr-x, the resonance
delocalization of electrons from para-aniline is expected to
decrease the aci-nitro character of the intermediate, reducing
the uncaging.19 The complex with Ca2+ ion weakens the
electron donation effect and thus, the presence of Ca2+
enhances the photolysis.
Figure 3. Emission spectra of fluo-3 (10 μM, excitation at 490 nm)
resulting from titration at 20 °C of nitr-T (1.0 mM in 40 mM Hepes/
100 mM KCI, pH 7.2) with 0.10 mM incremental additions of CaCl2.
Inset: Scatchard analysis (using a Kd value of 500 nM for fluo-3)21 of
these data.
Figure 4. UV−vis spectra of photolyzed nitr-T (20 μM in 40 mM
Hepes/100 mM KCI, pH 7.2) after titration with incremental
additions of CaCl2. Inset: Scatchard analysis of these data representing
the ratio between bound (B) and free (F) concentrations of the
photolyzed chelator nitr-T vs the concentration of free Ca2+. B/F =
[nitr-T]b/[nitr-T]f = (Amax − A)/(A − Amin), A = Absorbance at 360
nm.
Ca2+ Binding and Release. The affinity of nitr-T for Ca2+
ions was obtained by titrating nitr-T solutions with incremental
additions of Ca2+ using spectral10 and calorimetry techniques.
The concentration of the free Ca2+ ions [Ca2+]F after the
treatment of 1.0 mM solutions of nitr-T or nitr-S with various
amounts of CaCl2 was measured using the fluorescent dye fluo-
320 (see Experimental Section for details). In the absence of
calcium ions, the flu-3 has no fluorescence upon mono-
chromatic (λirr = 490 nm) illumination. The addition of CaCl2
triggers the emission of fluo-3 (λirr = 490 nm, λem = 530 nm)
and the fluorescence intensity is proportional to the calcium ion
concentration. Figure 3 shows the Ca2+ ion indicator
fluorescence of the fluo-3 added to the nitr-T-Ca2+ complex
solutions. From these data a value of Kd = 520 nM was
obtained (see Experimental Section and ref 10 for details). This
value is higher than the reported Kd of BAPTA (110 nM) or
nitr-5 (145 nM) determined by spectroscopic methods.1 The
lower affinity can be attributed to the weak electron-attracting
effect of the o-nitrobenzhydryl groups.
higher concentrations of free Ca2+ after exposure.1 The
photolyzed intermediate presents an affinity of 43 μM (Figure
S5 of the SI).
We measured the evolution of the free Ca2+ concentration in
irradiated nitr-T solutions using a calcium selective electrode.
Figure 5 presents these data as well as the calculated [Ca2+]F for
DM-nitrophen, NP-EGTA, nitr-5 and nitr-S. The concentration
of the free calcium ions jumped to a very high level at about
10% conversion in the case of the DM-nitrophen. NP-EGTA
also caused fairly large [Ca2+]F jumps at conversions up to
about 30%. In contrast, the changes in [Ca2+]F in the cases of
nitr-5 and nitr-S were small and continuous for all of the
conversion range. Compared to this, nitr-T provided small
changes at conversions lower than 50%, but it has shown strong
constant response for longer photolysis. The final calcium ion
concentration at full conversion almost reached the level of the
NP-EGTA or DM-nitrophen.
The affinity of the nitr-T photoproduct for Ca2+ was
estimated using UV−vis spectroscopy. Figure 4 presents the
titration curves of photolyzed nitr-T with CaCl2. Data analysis
gave a Kd of 1.6 mM for the photoproduct. It is important to
note that Kd of the photoproducts of nitr-5 (6.3 μM) and nitr-7
(3.0 μM) are significantly lower as determined by same
method.12 A low binding affinity of the photoproduct is a highly
desired property of phototriggerable Ca2+ cages, since it allows
As a consequence of the higher Kd of the chelator and lower
Kd of its photodegradation product, the affinity change of nitr-T
for Ca2+ ions upon photolysis (>3000-fold) is much higher than
the affinity change of nitr-5 (43-fold) or nitr-7 (54-fold).12 The
double substitution greatly improved the Ca2+-release and the
final concentration of free Ca2+ increased up to a level
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Figure 5. Changes in calcium ion concentration (pCa: −log[Ca2+]F)
versus photolysis conversion experimentally determined using a Ca2+-
electrode for nitr-T (triangles) and calculated (continuous lines) for
four different calcium cages (2 mM). The initial Ca2+ concentration
was 400 nM in all cases. [Ca2+]T values: 1.975 mM for DM-nitrophen,
1.667 mM for NP-EGTA, 1.468 mM for nitr-5, 1.404 mM for nitr-S,
and 0.870 mM for nitr-T.
Figure 6. The microcalorimetric titrations of nitr-T (A) and of its
photolytic product (B) in 40 mM HEPES (pH 7.2) containing 100
mM KCl. The raw data for the sequential injections of the 10 mM
CaCl2 into the 1 mM chelator are shown in the upper graphs, while
the integrated heats of complexation (black squares) and the fitting to
the correlation function (red lines) are depicted in the bottom panels.
comparable with EGTA-based cages. It is important to note
that a 1600-fold affinity change has been indicated for a doubly
substituted nitr- derivative in a review article,14 but it was never
validated by a scientific report. The Kd of nitr-S (170 nM) and
of its photoproduct (6.9 μM) were estimated using the same
methods (Figures S6 and S7 of the SI). An affinity change of
about 40-fold upon photolysis was obtained. These values are
close to those reported for nitr-5.12
Using the same titration method, the affinity of nitr-T for
Mg2+ ions (28 mM, Figure S8 of the SI) was determined. The
selectivity of nitr-T for Ca2+ against Mg2+ was 5 × 104 (Table
S1). The value is comparable to that of nitr-5 (6 × 104)12 and
slightly smaller than NP-EGTA (11 × 104).1
ITC Measurement. ITC was used to determine the main
thermodynamic parameters (enthalpy, entropy, affinity, stoi-
chiometry) of the bimolecular interaction in a single experi-
ment by measuring the heat change during the binding
event.22,23 Samples of nitr-T, nitr-S, and their photolysis
products were titrated with CaCl2 in buffered solutions (pH
7.2) at 25 °C. For comparison, EGTA and BAPTA were also
tested. A typical graphical representation of the titration is
shown in Figure 6A. Each addition of small portions of Ca2+
ions to the 1 mM solution of the BAPTA derivative gave an
endothermic thermal response, caused by the formation of the
complex between the chelator and the calcium ions. The
exchanged heat decreased after each injection because less and
less chelator molecules are available for complex formation. At
the end of the experiment, when the calcium ions are in high
excess and therefore most of the BAPTA is complexed, only the
CaCl2 dilution heat was detected.
The correlation of the raw data to a bimolecular interaction
fitting function indicated that the positive enthalpy of the
complex formation was compensated by a much higher
entropic contribution. This was a common feature for all of
the tested BAPTA derivatives (Table 1). In contrast, the Ca2+-
binding of EGTA induced a small positive entropic change and
a great enthalpy decrease, suggesting an enthalpy-driving
coordination.
photolytic products of nitr-T and nitr-S were similar to those of
nitr-T/S (Figure 6B). However, the detected affinities were
significantly lower (1.5 mM for the photolyzed nitr-T and 32.6
μM for the photolyzed nitr-S) than those of the parent
chelators. The calculated Ca2+ affinity loss of the nitr-T/S
derivatives upon light exposure was 428 fold for nitr-T and 27
fold for nitr-S (Table 1). The differences in the absolute values
of Kd obtained from the spectrometric and calorimetric
methods are attributed to the different experimental conditions
required for the experiments (i.e., different concentrations of
the nitr-T/S and of the CaCl2 in the tested solutions). In spite
of these differences, the observed trend in both cases confirmed
the significant benefit of the double substitution for light-
triggered Ca2+-release. The location of the strong electron-
withdrawing o-nitroso-benzoyl group in the para-position to the
amino-diacetate group decreased the electron density of the
nitrogen atom and consequently, dramatically influenced the
calcium binding properties of the molecule.
Ca2+ Release in Living Cells. The ability of nitr-T to
release Ca2+ in living cells was tested in patch clamp
experiments carried out in mouse chromaffin cells in primary
culture. We initially examined the resting Ca2+ levels at different
loading levels of intracellular Ca2+. Cells were patch clamped in
the whole cell recording mode. After a two minute equilibration
period, the cell was stimulated with a UV flash generated by a
flash lamp (Rapp Optoelectronics, Hamburg, Germany).
Immediately after the flash application, the cell was illuminated
with short (20 ms) illuminations at 350 and 380 nm at a rate of
4 Hz. This maintained the intracellular Ca2+ level near that
reached by the flash and allowed simultaneous ratiometric Ca2+
measurement. Figure 7A shows the basal free Ca2+ (filled
circles) and the free Ca2+ levels reached immediately after flash
photolysis (determined from the ratio of the first 350/380 nm
paired illumination, open circles) for a series of four different
Ca2+ loads in the patch solution. We did not increase the Ca2+
load above 4 mM because we wanted to keep the resting
intracellular Ca2+ below about 1.5 μM. The cells tolerated the
loading with Nitr-T quite well. Recordings of 40 to 60 min were
common with little degradation of the cell properties, which is
The presence of the electron withdrawing groups on the
aromatic rings caused a slight decrease of the Ca2+ ion affinity
of the BAPTA moiety (0.86 μM for BAPTA, 3.5 μM for nitr-T,
and 1.2 μM for nitr-S). The raw titration curves of the
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Table 1. Calcium Binding Parameters of the nitr-T/S Chelators and Their Photolytic Products As Determined by Means of ITC
and Spectroscopy
a
b
species
EGTA
N
ΔH kcal/mol
ΔS cal/mol·K -TΔS kcal/mol
ΔG kcal/mol
Kd mol·l‑1
417 × 10−9 ( 32 × 10−9
0.86 × 10−6 ( 0.11 × 10−6
)
3.5 × 10−6 ( 0.4 × 10−6
1.5 × 10−3 ( 0.2 × 10−3
1.2 × 10−6 ( 0.2 × 10−6
32.6 × 10−6 ( 3.4 × 10−6
Kd mol·l‑1
0.8
1.1
0.8
1.0
0.8
0.5
−6.94 ( 0.03)
2.32 ( 0.01)
2.36 ( 0.02)
4.05 ( 0.14)
2.10 ( 0.02)
2.64 ( 0.15)
5.90
35.5
−1.61
−9.69
−8.98
−7.24
−9.34
−8.76
−8.550 ( 0.03)
−7.67 ( 0.03)
−6.62 ( 0.03)
−3.19 ( 0.14)
−7.24 ( 0.02)
−6.12 ( 0.15)
)
150 × 10−9
110 × 10−9
520 × 10−9
1.6 × 10−3
170 × 10−9
6.9 × 10−6
BAPTA
nitr-T
32.9
24.5
34.2
29.4
)
)
)
)
photolyzed nitr-T
nitr-S
photolyzed nitr-S
a
b
Stoichiometry of the CaCl2:chelator complex The corresponding values for EGTA and BAPTA are reported in ref 7; the data for nitr-T, nitr-S and
for their photolytic products have been obtained by us from the spectroscopy-based determinations.
and at resting Ca2+ the releasable pools are quite small. For this
reason, we generally load NP-EGTA to generate free calcium in
the range of 500−700 nM, which leads to increased photolysis
induced catecholamine release. Typically, loading to about 80%
of added NP-EGTA (e.g., 4 mM Ca2+ and 5 mM NP-EGTA)
produces cytosolic free Ca2+ near 350 nM. Using nitr-T, we
reached acceptable resting free Ca2+ levels with up to 2.5 mM
Ca2+ added to 6.5 mM nitr-T, (free Ca2+ typically near 700−
800 nM). In experiments requiring a resting (prephotolysis)
calcium concentration closer to the physiological resting level in
most cells (50−250 nM), less calcium may be added, and
therefore less nitr-T will be loaded with Ca2+, due to its
relatively low prephotolysis Ca2+ affinity. Under these
conditions, photolysis of nitr-T will result in smaller increases
in free Ca2+, limiting its efficacy.
Ca2+ released by flash photolysis effectively elicited vesicle
fusion in chromaffin cells loaded with nitr-T (Figure 8A).
Figure 7. (A) The relationship between Ca2+ loading and free Ca2+ in
nitr-T loaded cells. Chromaffin cells were patch clamped using an
intracellular solution containing 6.25 mM nitr-T. This solution was
complemented with added Ca2+ stock to reach nominal Ca2+
concentrations of 2 to 4 mM. The free [Ca2+]i after equilibration
(solid circles) the free [Ca2+]i reached after flash photolysis under our
normal experimental conditions (open circles) are plotted versus the
nominal intracellular Ca2+. The free Ca2+ in both cases is dependent
on the loading of nitr-T. (B) Ca2+ concentrations reached after
photolysis are dependent on the resting free Ca2+. The dependence on
the [Ca2+]i reached after flash photolysis on the preflash [Ca2+]i is
shown for cells recorded after loading as described in the text.
Figure 8. (A) Membrane capacitance, recorded by patch clamp in the
whole cell configuration (solid line) following flash photolysis of nitr-
T. The free [Ca2+]i (solid circles) is shown prior to and after flash
photolysis. (B) Monochromator elicited Ca2+ ramp stimulation also
drives membrane fusion by photolysis of nitr-T. In patch clamped
chromaffin cells loaded with nitr-T as described above, application of
trains of alternating illumination at 350 and 380 nm (20 ms duration)
at 4 Hz slowly raised free [Ca2+]i (squares) and caused fusion of
vesicles, resulting in a capacitance increase (solid line).
much longer than the duration of our typical flash experiment.
The amount of Ca2+ release was dependent on the degree of
Ca2+ loading of nitr-T. Figure 7B is a scatter plot which shows
the relationship between the basal free [Ca2+]i and the response
to the UV flash illumination. Under our conditions, we do not
reach the upper ranges of photolytic release capacity due to
relatively low loading of the nitr-T with Ca2+. In spite of this
low loading, we see increases of Ca2+ to low μM concentrations
with 2 mM Ca2+ added to 6.25 mM nitr-T. At 3−4 mM added
Ca2+, calcium release can approach 10 to 20 μM, depending on
the conditions. Thus, these experiments show the successful
release of Ca2+ from nitr-T in living cells. Higher Ca2+ loading
would result in unacceptably high [Ca2+]i. However, at lower
added Ca2+ (∼35% saturation) we were still able to generate
appreciable increases in [Ca2+]i. The resting free [Ca2+] in
chromaffin cells is near 100 nM. Priming is calcium sensitive
Exocytosis of large dense cored vesicles, as tracked by
membrane capacitance changes, demonstrated that photolyzed
Ca2+ is available to elicit Ca2+ dependent cellular processes.
Membrane capacitance, recorded by patch clamp in the whole
cell configuration rose significantly following flash photolysis of
nitr-T.
We have also applied trains of alternating 350 and 380 nm
illuminations utilizing the monochromator, to slowly photo-
lytically release Ca2+ without flash application (ramp
stimulation) from nitr-T (with simultaneous monitoring of
free Ca2+) and found that this method also generates effective
Ca2+ release which also drove vesicle fusion with the plasma
membrane. Figure 8B shows one such experiment in which a
chromaffin cell with a baseline Ca2+ near 400 nM was
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illuminated at 4 Hz for 12 s. The intracellular Ca2+ (filled
squares) rose during the train of illuminations and as it
approached 1 μM the membrane capacitance (solid line) began
to rise, indicating fusion of vesicles with the plasma membrane.
Thus nitr-T can also release Ca2+ under more moderate UV
illumination.
1,2-Bis{2-amino-5-[(5-methoxy-2-nitrophenyl)hydroxymethyl]-
phenoxy}ethane-N,N,N′,N′-tetraacetic Acid Tetraethyl Ester (Nitr-T
Tetraethyl Ester). In a dark environment, trimethylsilyl trifluorome-
thanesulfonate (TMS OTf) (0.544 mL, 2.72 mmol) was added
dropwise into a solution of BAPTA tetraethyl ester (160 mg, 0.272
mmol), 5-methoxy-2-nitrobenzaldehyde (130 mg, 0.72 mmol), and
2,6-di-tert-butylpyridine (0.72 mL, 3.26 mmol) in dry CH2Cl2 (4 mL)
at room temperature under inert atmosphere. TLC and 1H NMR
suggested the reaction finished after stirring in dark environment for
72 h. The reaction solution was diluted with CH2Cl2 (10 mL), poured
into saturated aqueous NaHCO3 (50 mL) and the organic phase was
then separated. The aqueous layer was extracted with CH2Cl2. The
combined extracts were washed with water, dried over Mg2SO4, and
evaporated to dryness to yield a yellow oil. The oil was dissolved in
CHCl3 (30 mL) and tetrabutylammonium fluoride trihydrate (260 mg,
0.82 mmol) was added. After 30 min stirring in a dark environment at
room temperature, the solution was evaporated to dryness, following
by dissolved in ethyl acetate-toluene (1:1 v/v, 100 mL), washed with
water, dried over Mg2SO4, and the solvent was evaporated to yield the
crude product as orange oil. The crude product was purified
immediately by silica flash column with ethyl acetate−hexane (1:1)
as eluant (Rf: 0.4) afforded the pure product (238 mg, yellow solid,
CONCLUSIONS
■
The properties of doubly functionalized nitr-T reported in this
manuscript demonstrate that nitr-T is a potential new Ca2+ ion
cage for cell biology with greatly improved release efficiency
against the singly substituted nitr-x chelators, while maintaining
their pH insensitivity in the physiological range and the high
Ca2+/Mg2+ selectivity. Our patch clamp experiments show that
nitr-T is well tolerated by the living cells. Moderate Ca2+ jumps
were generated using exposure conditions compatible with sub
μM resting [Ca2+]. At low amounts of the added Ca2+ (∼35%
loading), we were able to generate appreciable increases in
[Ca2+]i. Under conditions where higher resting [Ca2+]i can be
tolerated, larger Ca2+ jumps could be generated. Further
improvement of the releasing and photolytic properties of the
doubly functonalized nitr-T by using other chromophores
attached to the BAPTA ring can be envisioned and are
currently in progress in our group.
1
yield 92%). The product was immediately used for the next step. H
NMR (250 MHz, CDCl3, δ ppm): 8.08−8.04 (dd, 2H, aromatic),
7.36−7.34 (t, 2H, aromatic), 6.91−6.86 (m, 4H, aromatic), 6.73−6.68
(m, 4H, aromatic), 6.46 (s, 2H, ArCH), 4.21−4.19 (d, 4H,
OCH2CH2O), 4.10 (s, 8H, NCH2), 4.07−3.96 (m, 8H, OCH2),
3.91 (s, 3H, OCH3), 1.16−1.10 (m, 12H, CH2CH3). 13C NMR (62.5
MHz, CDCl3, δ ppm): 171.53 (CO), 163.68, 149.93, 142.19,
140.76, 139.07, 135.67, 127.90, 120.02, 118.35, 114.03, 112.88, 112.63
(12C, aromatic), 71.42 (ArCH), 67.11 (OCH2CH2O), 60.89 (OCH2),
55.94 (OCH3), 53.44 (NCH2), 14.00 (CH2CH3). MS
(C46H56N4O182+): 952.6 (Calculated 952.9).
EXPERIMENTAL SECTION
■
Materials and Methods. 5-Hydroxy-2-nitro-benzaldehyde (97%,
Sigma-Aldrich), iodomethane (99%, Sigma-Aldrich), trimethylsilyl
trifluoromethanesulfonate (TMS OTf, 98%, Sigma-Aldrich), 2,6-di-
tert-butylpyridine (97%, Sigma-Aldrich), BAPTA tetraethyl ester (98%,
Nitr-S Tetraethylester. An analogous method was used to
synthesize the nitr-S, with the difference that the molar ratio of
BAPTA tetraethyl ester: 5-methoxy-2-nitrobenzaldehyde: TMS OTf
Alfa), KCl (99%, Ca2+ < 0.001%, Riedel-de Haen), and solvents
̈
max
were used as purchased. Preparative column chromatography and flash
column chromatography were carried out using silica gel (60 Å pore
size, 63−200 μm particle size) from MerckK GaA (Darmstadt,Ger-
many).
1
was 1:1:1. Yield: 50%. H NMR (250 MHz, CDCl3, δ ppm): 8.00−
7.96 (d, 1H, aromatic), 7.23−7.22 (d, 1H, aromatic), 6.82−6.72 (m,
6H, aromatic), 6.69−6.60 (m, 2H, aromatic), 6.38 (s, 2H, ArCH), 4.17
(s, 4H, OCH2CH2O), 4.06 (s, 4H, NCH2), 4.05(s, 4H, NCH2), 3.99−
3.91 (m, 8H, OCH2), 3.91 (s, 3H, OCH3), 1.08−1.03 (m, 12H,
CH2CH3).
Solution 1H and 13C spectra were measured in CDCl3 or DMSO-d6
solution at 25 °C with a Bruker Ultra Shield 250 MHz spectrometer.
UV spectra were recorded on a Varian Cary 4000 UV−vis
spectrometer (Varian Inc. Palo Alto, CA). Fluorescence spectra were
obtained from TIDA S400 fluorescence spectrometer (J&M,
Germany). Mass spectra were recorded on a Micromass Finnigan-
MAT ZAB-HS mass spectrometer. A calcium ion-selective electrode
(Orion type 9720BNWP, Pulse Instruments, U.S.) was used for
determining the free Ca2+ concentrations. HPLC analysis and
purification of the compounds were performed with a JASCO
HPLC 2000 (Jasco, Groß-Umstadt, Germany) equipped with a
diode array UV−vis detector and fraction collector. Reprosil 100 C18
columns were used for preparative (250 × 20 mm) and analytical (250
× 5 mm). Water containing 0.1% TFA and acetonitrile containing 5%
water and 0.1% TFA were used as eluents. Analytical and preparative
runs were performed at a solvent flow of 1 mL/min and 10 mL/min,
respectively, and by increasing the volume fraction of acetonitrile after
a 3 min run from 0% to 100% within 30 min and successively keeping
this ratio for another 7 min.
1,2-Bis{2-amino-5-[(5-methoxy-2-nitrophenyl)hydroxymethyl]-
phenoxy}ethane-N,N,N′,N′-tetraacetic Acid (Nitr-T). Nitr-T tetraeth-
yl ester (200 mg, 0.21 mmol) was dissolved in a mixture of methanol
(10 mL) and 1,4-dioxane (5 mL) and then saponified by the addition
of an excess of 1 M aqueous NaOH and stirring overnight at room
temperature. After acidification with HCl aqueous to pH 3, the
precipitate was collected and dried to afford the product (169 mg,
1
yellow solid). Yield: 96%. H NMR (250 MHz, DMSO-d6, δ ppm):
8.02−7.98 (dd, 2H, aromatic), 7.40−7.39 (d, 2H, aromatic), 7.06−
7.05 (d, 2H, aromatic), 6.94 (s, 2H, aromatic),6.67−6.63 (m, 4H,
aromatic), 6.29 (s, 2H, ArCH), 4.21 (s, 4H, OCH2CH2O), 4.02 (s,
8H, NCH2), 3.88 (s, 6H, OCH3). 13C NMR (62.5 MHz, CDCl3, δ
ppm): 172.80 (CO), 162.96, 149.02, 143.10, 140.44, 138.10, 135.83,
127.22, 119.80, 117.69, 113.61, 113.49, 112.72 (12C, aromatic), 69.24
(ArCH), 67.19 (OCH2CH2O), 55.96 (OCH3), 53.44 (NCH2). MS
(C38H38N4O18Na+): 861 (Calculated 861.2). Elemental Anal. Calcu-
lated for C38H38N4O18: C, 54.42; H, 4.57; N, 6.68. Found: C, 54.19; H,
4.39; N, 6.59.
1-{2-Amino-5-[(5-methoxy-2-nitrophenyl)hydroxymethyl]-
phenoxy}-2-(2-amino-phenoxy)ethane-N,N,N′,N′-tetraacetic Acid
(Nitr-S). Yield: 96%. 1H NMR (250 MHz, DMSO-d6, δ ppm):
8.01−7.98 (d, 1H, aromatic), 7.39−7.38 (d, 1H, aromatic), 7.06−6.75
(m, 6H, aromatic), 6.65−6.61 (m, 2H, aromatic), 6.28 (s, 1H, ArCH),
4.23 (s, 4H, OCH2CH2O), 4.04 (s, 4H, NCH2), 4.01(s, 4H, NCH2),
3.88 (s, 3H, OCH3). MS (C30H31N3O14Na+): 680 (Calcd. 680.2).
Anal. Calcd. for C30H31N3O14: C, 54.80; H, 4.75; N, 6.39. Found: C,
54.61; H, 4.59; N, 6.21.
Synthesis. 5-Methoxy-2-nitrobenzaldehyde.24 A mixture of 5-
hydroxy-2-nitro-benzaldehyde (1.0 g, 6 mmol), iodomethane (0.75
mL, 12 mmol), and potassium carbonate (4.13 g, 30 mmol) in 50 mL
of DMF was stirred for 12 h at rt. The resulting yellow suspension was
filtered and the solid was washed with dichloromethane. The filtrate
and washing solutions were combined and dried over Na2SO4. The
organic solvent was removed under reduced pressure and the solid was
recrystallized from a mixture of ethyl acetate/petroleum ether to yield
1
0.73 g yellow needle crystal solid. Yield: 67%. H NMR (250 MHz,
CDCl3, δ ppm): 10.41 (s, H, CHO), 8.10−8.06 (d, H, aromatic),
7.26−7.24 (d, H, aromatic), 7.09−7.05 (dd, H, aromatic), 3.87 (s, 3H,
OCH3).
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Journal of the American Chemical Society
Article
1,2-Bis{2-amino-5-[(5-methoxy-2-nitrosophenyl)carbonyl]-
phenoxy}ethane-N,N,N′,N′-tetraacetic Acid (Final Photolysis Prod-
uct of Nitr-T). A solution of nitr-T in DMSO (1 mL, 10 mg/mL) was
irradiated under 360 nm with 180 J/cm2 and then the photoproduct
was isolated with preparative HPLC. After removed the solution by
freeze-drying, 1.8 mg yellow powder was obtained. The product was
used immediately. 1H NMR (250 MHz, DMSO-d6, δ ppm): 7.65−7.62
(dd, 2H, aromatic), 7.50−7.49 (d, 2H, aromatic), 7.29−7.25 (dd, 2H,
aromatic), 7.19−7.18 (d, 2H, aromatic),6.97−6.93 (dd, 2H, aromatic),
6.54−6.50 (d, 2H, aromatic), 4.30 (s, 4H, OCH2CH2O), 4.15 (s, 8H,
NCH2), 3.99 (s, 6H, OCH3). MS (C38H34N4O16Na+): 825 (Calcd.
825.2). HPLC analysis suggested a purity of 97%.
This method was also used to calculate the affinity of chelator-Mg2+
before irradiation under similar condition. The concentration of
photoproducts (photolyzed nitr-T/S) used was 10 μM. The solutions
were buffered to pH 7.2 with 40 mM Hepes and the ionic strength was
set with 100 mM KCl.
Experimental Determination of the Free Ca2+ Using the Ion-
Selective Calcium Electrode. The calcium electrode was calibrated
at 20 °C using a 0.1 M CaCl2 calibration standard solution diluted to
concentrations between 1 × 10−2 M, and 4 × 10−7 M. A sample
containing 2 mM nitr-T and 0.87 mM Ca2+ was used for the
experiments (data in Figure 5) and irradiated for various times. The
concentration of the free Ca2+ was determined by interpolating the
measured values in the calibration curve. The corresponding
1-{2-Amino-5-[(5-methoxy-2-nitrosophenyl)carbonyl]phenoxy}-
2-{2-Amino-5-[(5-methoxy-2-nitrophenyl)hydroxymethyl]phenoxy}-
ethane-N,N,N′,N′-tetraacetic Acid (Photolysis Intermediate). A
solution of nitr-T in DMSO (2 mL, 10 mg/mL) was irradiated
under 360 nm with 100 J/cm2 and then isolated as photolyzed nitr-T.
0.6 mg yellow powder was obtained after HPLC purification. The
1
conversion of nitr-T for each irradiation time was calculated by H
NMR spectroscopy.
Isothermal Titration Calorimetry (ITC). Titration calorimetry
measurements were performed with a VP-ITC calorimeter (Microcal,
Northampton, MA). In a typical experiment, 27 aliquots of 10 μL
buffered (40 mM HEPES pH 7.2 containing 100 mM KCl) solution of
3.3 mM CaCl2 were added into the measuring cell containing 1.4 mL
of the calcium chelator (330 μM in the same buffer) using a 250 μL
rotating syringe at constant temperature (25 °C). The gap between
two consecutive injections was set to 10 min, in order to allow the
system to reach the thermodynamic equilibrium between injections.
The heat change associated with the dilution of the CaCl2 into buffer
was determined (control experiments), using the same number of
injections and the same CaCl2 concentration as employed in the
titration experiment. The heat of interaction for each injection was
measured by the integration of each titration peak using the ORIGIN
7 software (OriginLab Co. Northampton, MA, delivered with the VP-
ITC). For each injection, the dilution heats in the control experiment
were subtracted from the heat obtained in the interaction experiment.
The resulting corrected curves with the heat of the interaction were
used for the calculations of the stoichiometry, molar enthalpy,
equilibrium constant, entropy, and Gibbs free energy of the
complexation. The thermodynamic parameters were determined by
applying the correlation function corresponding to one binding site
model. The experiments were performed using nitr-T, nitr-S and their
photolysis products after HPLC purification.
1
product was used immediately. H NMR (250 MHz, DMSO-d6, δ
ppm): 8.01−7.98 (d, 1H, aromatic), 7.68−7.63 (dd, 1H, aromatic),
7.50 (s, 1H, aromatic), 7.39−7.37 (d, 1H, aromatic), 7.30−7.25 (dd,
1H, aromatic), 7.17−7.16 (d, 1H, aromatic), 7.06−7.01 (dd, 1H,
aromatic), 6.96−6.94 (m, 2H, aromatic), 6.68−6.60 (m, 2H,
aromatic), 6.58−6.52 (t, 1H, aromatic), 6.29 (s, 1H, ArCH), 4.29−
4.20 (t, 4H, OCH2CH2O), 4.16 (s, 4H, NCH2), 4.03 (s, 4H, NCH2),
3.99 (s, 3H, OCH3), 3.88 (s, 3H, OCH3). MS (C38H36N4O17Na+):
843 (Calcd. 843.2). HPLC analysis suggested a purity of 93%.
Irradiation Conditions and Quantum Efficiency. A LUMOS
43 lamp (Atlas Photonics) equipped with a LED at 360 nm was used
for the experiments. The quantum flow of the lap was 2.77 × 10−9 E/s
as measured by ferrioxalate actinometry. For the evaluation of the
quantum yield of the photouncaging reaction, the number of reacted
molecules (i.e., the conversion) for a given exposure time (i.e., the
number of photons) was determined by 1H NMR and UV
spectroscopy for conversions below 10%. For these experiments 2.5
mL of a 6 mM solution of either nitr-T or nitr-S in Na2HPO4/
NaHPO4 buffer (in D2O, 0.044 wt % of NaHPO4 and mol 0.18 wt %
Na2HPO4) at pH 7.2 was used and various portions of 17 mM CaCl2
were added to each aforementioned solution in the case of the nitr-T/
S−Ca2+ complexes photolysis experiments.
Evaluation of Ca2+ Release in Living Cells. Calcium release
from nitr-T in living cells was tested under quasi-physiological
conditions. nitr-T was applied as a component of the intracellular
solution in patch clamp experiments carried out in mouse chromaffin
cells in primary culture. The nitr-T was dissolved in 20 mM HEPES/
CsOH solution. The resulting 20 mM nitr-T-Hepes solution was
added to Cs-glutamate to reach a final concentration of 6.25 mM nitr-
T and 6.25 mM Hepes. The intracellular solution also contained 2 mM
MgATP, 0.5 mM Na-GTP, 200 μM MagFura and Fura4F, and 2−4
mM CaCl2. The final pH was 7.32 and the final osmolarity was 291
mOsm.
Whole cell recordings were carried out in mouse chromaffin cells.
The methods for cell preparation and experimental procedures have
been previously described.25 A monochromator (Till Photonics,
Planegg, Germany) was used to induce photolysis by illumination at
350 and 380 nm in ramp experiments, which allowed simultaneous
ratiometric determination of the free intracellular calcium, by
extrapolation to an in vivo calibration curve generated for the dye
mixture.26 In addition to monitoring changes in the ratiometrically
determined intracellular free calcium, we used increases in membrane
capacitance as an independent readout for the efficacy of released
calcium to trigger membrane fusion of large dense core granules.
Ca2+ Binding Affinity. The stability constant for the equilibrium
binding of Ca2+ to the chelators nitr-T or nitr-S (Ka) was calculated
using the method of Ellis-Davies et al.10 by the equation:
Ka([nitr − T/S]T − [Ca2+]B) = [Ca2+]B /[Ca2+
]
F
where [Ca2+]F is the concentrations of the free Ca2+, [Ca2+]B is the
concentration of Ca2+ bound to the chelator, and [nitr-T]T or [nitr-S]T
the total concentration of the chelator in the solution. The values of
[Ca2+]F (and hence [Ca2+]B) were obtained by measuring the emission
of a calcium-sensitive dye (fluo-3, Kd = 500 nM) during the titration
with 0.10 mM CaCl2.8
In this experiment, 1.0 mM solutions of the chelators in 40 mM
Hepes pH 7.2 + 100 mM KCl have been used.
After the full light exposure, the estimates of [Ca2+]B/[Ca2+]F were
obtained from the absorption curves by the method described
previously.12 The concentration of nitr-T used for irradiation was 20
μM and the concentrations of CaCl2 were 0.04, 0.40, 1.71, 6.85, 15.0,
and 240 mM, respectively (further increase the concentration did not
change the absorption curve any more). The [Ca2+]F is close to
[Ca2+]T due to the fact that the concentration of the calcium ions is
significantly higher that the chelators concentrations. The [Ca2+]B (=
[chelator]B) was obtained from the UV−vis spectra by measuring the
absorbance of the chelator solutions in the presence and in the absence
of CaCl2. The equation:
ASSOCIATED CONTENT
■
S
[chelator]B /[chelator]F = (Amax − Ax)/(Ax − Amin
)
* Supporting Information
1
UV spectra, fluorescence spectra, H NMR spectra, and the
(where Amax is the absorbance of the chelator without Ca2+, Amin is the
absorbance of the chelator in the presence of a high excess of Ca2+, and
Ax is the absorbance with setted Ca2+ concentration) gave the
complexed chelator concentration.
table collecting the properties of calcium caged compounds.
This material is available free of charge via the Internet at
http://pubs.acs.org.
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Journal of the American Chemical Society
Article
Lehmann, H.; Kalb, O.; Kneissel, M.; Missbach, M.; Muller, I. R.;
̈
AUTHOR INFORMATION
Corresponding Author
delcampo@mpip-mainz.mpg.de
■
Reidemeister, S.; Renaud, J.; Taillardat, A.; Tommasi, R.; Weiler, S.;
Wolf, R. M.; Seuwen, K. J. Med. Chem. 2010, 53, 2250−2263.
(25) Liu, Y.; Schirra, C.; Stevens, D.; Matti, U.; Speidel, D.; Hof, D.;
Bruns, D.; Brose, N.; Rettig, J. J. Neurosci. 2008, 28, 5594−5601.
(26) Thomas, V. Neuron 2000, 28, 537−545.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
A.d.C., J.C., and R.A.G. acknowledge financial support from the
DFG (Projects CA880/3-1) and from the Materials World
Network Program (DFG AOBJ 569628).
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̀
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