Optical control of calcium affinity in a spiroamido-rhodamine based calcium chelator

Article abstract of DOI:10.1021/ol200408j

An optically controlled Ca²⁺-chelator 1 was developed to mimic natural calcium oscillations. Compound 1, a spiroamido-rhodamine derivative of 1,2-bis(o-aminophenoxy)ethane-N,N,N0,N0-tetraacetic acid (BAPTA), underwent cycles of reversible trans

Full text of DOI:10.1021/ol200408j

ORGANIC
LETTERS
2011
Vol. 13, No. 8
2018–2021
Optical Control of Calcium Affinity in a
Spiroamido-rhodamine Based Calcium
Chelator
Liangxing Wu, Yingrui Dai, and Gerard Marriott*
Department of Bioengineering, University of California;Berkeley, Berkeley, California
94720, United States
marriott1@berkeley.edu
Received February 14, 2011
ABSTRACT
An optically controlled Ca2þ-chelator 1 was developed to mimic natural calcium oscillations. Compound 1, a spiroamido-rhodamine derivative of
1,2-bis(o-aminophenoxy)ethane-N,N,N0,N0-tetraacetic acid (BAPTA), underwent cycles of reversible transitions between a colorless closed state
and a fluorescent open form. The closed-state exhibited a high affinity for Ca2þ (Kd: 509 nM) with excellent selectivity over Mg2þ (Kd: 19 mM). The
open isomer had a 350-fold lower Ca2þ affinity (Kd: 181 μM), while the Mg2þ affinity was not significantly affected (Kd: 14 mM).
Oscillations in the intracellular calcium (Ca2þ) concen-
tration exist in many types of cells and are believed to
regulate a variety of physiological functions.1 The ability to
artificially generate similar calcium oscillations in cells
would be a valuable tool to understand the physiological
role of the oscillatory calcium signals and to study the
Ca2þ-dependent biological processes.2 Photochromic
probes that reversibly bind and release Ca2þ in response
to light are being used as part of this approach. To be
biologically useful, such probes should also fulfill the
following requirements:3 (1) Since the free Ca2þ concen-
tration in most resting cells is in a submicromolar range,
the resting state of the probe should bind Ca2þ with a
similar or lower dissociation constant, in order to store a
significant amount of Ca2þ at equilibrium. (2) The two
states of the probe should have at least a 10-fold difference
in binding affinity for Ca2þ. (3) Because the typical
intracellular free Mg2þ concentration is around mM, then
in order to avoid saturating by Mg2þ in cells, the probe
should exhibit excellent selectivity for Ca2þ over Mg2þ. (4)
The required wavelength for photochemistry should not
damage living cells; therefore, the wavelengths should be at
least above 300 nm. (5) The probe should be switchable in
an aqueous environment. (6) One of the two states of the
probe should incorporate a fluorescence readout allowing
the user to determine the location of the switch and to
monitor the release of Ca2þ in the sample.
Several attempts have been made to develop photore-
versible Ca2þ chelators.4 However, the reported probes are
still far away from satisfying the criteria detailed above.
Two favored photochemical reactions, such as geometric
isomerization of azobenzene or spiropyran to sterically
disrupt the binding pocket and ring-opening of spiropyran
to generate additional binding site, have been employed to
provide a means to optically control the Ca2þ binding
(4) (a) Kumar, S.; Hernandez, D.; Hoa, B.; Lee, Y.; Yang, J. S.;
McCurdy, A. Org. Lett. 2008, 10, 3761–3764. (b) Kumar, S.; Chau, C.;
Chau, G.; McCurdy, A. Tetrahedron 2008, 64, 7097–7105. (c) Momo-
take, A.; Arai, T. Tetrahedron Lett. 2003, 44, 7277–7280. (d) Sakata, T.;
Jackson, D. K.; Mao, S.; Marriott, G. J. Org. Chem. 2008, 73, 227–233.
(e) Filley, J.; Ibrahim, M. A.; Nimlos, M. R.; Watt, A. S.; Blake, D. M. J.
Photochem. Photobiol. A 1998, 117, 193–198. (f) Roxburgh, C. J.;
Sammes, P. G. Eur. J. Org. Chem. 2006, 1050–1056.
(1) (a) Fewtrell, C. Annu. Rev. Physiol. 1993, 55, 427–454. (b) Tsien,
R. W.; Tsien, R. Y. Annu. Rev. Cell Biol. 1990, 6, 715–760. (c) Thul, R.;
Bellamy, T. C.; Roderick, H. L.; Bootman, M. D.; Coombes, S. Adv.
Exp. Med. Biol. 2008, 641, 1–27.
(2) Dolmetsch, R. E.; Xu, K.; Lewis, R. S. Nature 1998, 392, 933–936.
(3) Ellis-Davies, G. C. R. Methods Enzymol. 2003, 360, 226–238.
r
10.1021/ol200408j
Published on Web 03/21/2011
2011 American Chemical Society

Figure 1. (a) Design and (b) synthesis of rhodamine-based Ca2þ chelator 1; (c) a model compound 2 for the open form of 1.
affinity. Such mechanisms suffer from the problem that
Ca2þ will stabilize the isomer that binds it, thus the photo-
chemistry will be hindered by the Ca2þ binding. Moreover,
none of the probes has good Ca2þ/Mg2þ selectivity.
photoinduced ring-opening reaction generates the rhoda-
mine chromophore, which significantly decreases the elec-
tron density in the BAPTA part and thus produces the
desired drop in Ca2þ affinity. The open isomer reverts
thermally to the closed form with a characteristic lifetime
of a few milliseconds in polar solvents,5 restoring the
affinity for Ca2þ. The design strategy proves to work well,
though further optimization is still needed to improve the
photochromic properties of the scaffold.
The synthesis of the reversible Ca2þ chelator 1 is sum-
marized in Figure 1b. The key starting fragments 3 and 4
were prepared according to reported procedures7,8 with
slight modifications. Condensation of 3 with 4 was con-
ducted in neat TFA at elevated temperature to afford
rhodamine 5, which was isolated as the colorless ring-
closed isomer. The spirolacton form was further confirmed
by the characteristic carbon signal near 84 ppm in 13C
NMR spectrum.5 Target compound 1 was obtained by
amidation of rhodamine 5 with propylamine followed by
hydrolysis of the four ethyl esters in the BAPTA part. To
estimate the Ca2þ binding affinity of the short-lived open
form of chelator 1, a model compound 29 (Figure 1c),
which is structurally and electronically similar to the open
form of 1 and exists only in the open state, was also
designed. Rhodamine 2 was synthesized from intermediate
Herein, we report the design, synthesis and properties of
the first photoreversible Ca2þ chelator 1 (Figure 1a) that
can satisfy all the aforementioned requirements. Our
approach to the design of 1 is displayed in Figure 1a.
The novel photoreversible calcium chelator 1 is based on a
photochromic rhodamine scaffold5 and a Ca2þ-chelating
moiety 1,2-bis(o-aminophenoxy)ethane-N,N,N0,N0-tetraa-
ceticacid(BAPTA).6 Thewell-known Ca2þ-chelatorBAP-
TA is chosen as the starting point because of its high Ca2þ
affinity and excellent Ca2þ/Mg2þ selectivity. Moreover, its
Ca2þ affinity can be easily adjusted by modulating the
substitutions on its benzene rings. Electron-withdrawing
or -donating substituents will decrease or increase the
affinity for Ca2þ, respectively. These properties have been
usedtodevelop caged calcium complexes.6b However, cage
compounds are less suitable for the study of oscillatory
calcium signals due to the irreversible photochemical
reactions. Reversible modulation of the electron density
of the BAPTA moiety would lead to release or bind Ca2þ
without steric disruption of the binding site. A suitable
photochromic reaction is envisioned to be the well-estab-
lished transformation between the closed and open form of
rhodamine amide derivatives5 as shown in Figure 1a. The
(7) Liu, Q.-H.; Yan, X.-L.; Guo, J.-C.; Wang, D.-H.; Li, L.; Yan, F.-
Y.; Chen, L.-G. Spectrochim. Acta, Part A 2009, 73A, 789–793.
(8) Grynkiewicz, G.; Poenie, M.; Tsien, R. Y. J. Biol. Chem. 1985,
260, 3440–3450.
(9) Similar compounds based on rhodol and BAPTA were developed
by Clarke and co-workers for ratiometric imaging of calcium ion. For
details, see: Simth, G. A.; Metcalfe, J. C.; Clarke, S. D. J. Chem. Soc.,
Perkin Trans. 2 1993, 1195–1204.
(5) (a) Foelling, J.; Belov, V.; Kunetsky, R.; Medda, R.; Schoenle, A.;
Egner, A.; Eggeling, C.; Bossi, M.; Hell, S. W. Angew. Chem., Int. Ed.
2007, 46, 6266–6270. (b) Knauer, K. H.; Gleiter, R. Angew. Chem. 1977,
89, 116–117.
(6) (a) Tsien, R. Y. Biochemistry 1980, 19, 2396–2404. (b) Adams,
S. R.; Kao, J. P. Y.; Grynkiewicz, G.; Minta, A.; Tsien, R. Y. J. Am.
Chem. Soc. 1988, 110, 3212–3220.
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5 using similar conditionsasdescribed for compound 1 (see
the Supporting Information for details).
form of 1 with Mg2þ were analyzed analoguously
(Supporting Information, Figure S3). The effect of Mg2þ
binding on the absorption spectrum was much smaller
compared with the effect of Ca2þ, presumably because
Mg2þ binds mainly to just one half of the chelator and does
not perturb the spectrum of the other parts.6,8 The Mg2þ
dissociation constant for 1 was found to be 19 mM with a
1:1 binding stoichiometry. Thus, the closed form of 1 has
high affinity for Ca2þ and exhibits excellent selectivity for
Ca2þ over Mg2þ
.
Figure 2. (a) Absorption spectra of 1 (20 μM) as a function of
[Ca2þ
]free (10 mM MOPS, 100 mM KCl, pH 7.2). Further
increase in [Ca2þ
]free after 39 μM had little effect on the spectrum
indicating complete saturation of 1 by Ca2þ was achieved. (b)
Hill plot of the absorbance measured at 320 nm.
Rhodamine-based chelator 1 dissolved freely in aqueous
solution at physiological pH. UVꢀvis absorption spectra
of 1 at various levels of free Ca2þ in MOPS buffer at pH 7.2
were displayed in Figure 2a. In the absence of Ca2þ, the
spectrum showed a maximum at 240 nm (ε: 34200
Mꢀ1 cmꢀ1) with a shoulder at 320 nm (ε: 5600 Mꢀ1 cmꢀ1).
Figure 3. (a) Emission spectra of 2 (2 μM, λex = 575 nm) as a
function of [Ca2þ
free (10 mM MOPS, 100 mM KCl, pH 7.2).
]
Further increase in [Ca2þ
]free after 1.68 mM had little effect on
the spectrum indicating complete saturation of 2 by Ca2þ was
achieved. (b) Hill plot of the emission measured at 600 nm.
3
3
There was no absorbance in the visible range, indicating
that compound 1 only existed as the closed form under
such conditions. The closed form was practically nonfluor-
escent. Addition of Ca2þ to the solution did not result in
any detectable formation of the open isomer. Small but
reproducible absorption changes in the UV region were
observed upon Ca2þ binding: the peak at 240 nm was
slightly blue-shifted, and the absorbance at 320 nm de-
creased with addition of Ca2þ. The titrationswere repeated
three times, and the data were analyzed by a Hill plot6a
(Figure 2b), i.e., a plot oflog [(A ꢀ Amin)/(Amax ꢀ A)] vslog
The open form of 1 is short-lived,5 preventing the direct
measurement of the binding affinities. Thus, a model
compound 2 was used to estimate the dissociation con-
stants of the metal complex of 1-open. Compound 2
absorbs maximally at 575 nm (ε: 98000 Mꢀ1 cmꢀ1) and
3
emits at around 600 nm, which are pretty much as expected
for rhodamine derivatives. Figure 3a shows the fluores-
cence spectra of 2 at different Ca2þ concentrations. The
fluorescence intensities were enhanced by addition of
Ca2þ. However, Ca2þ binding caused little change in the
wavelength of emission. The titration data were fitted to
Hill plot to give an apparent Kd of 181 μM with a 1: 1
binding for Ca2þ. The affinity for Ca2þ was 350-fold lower
than that of the closed form of 1, indicating the success of
our design strategy. The binding of 2 with Ca2þ reduced
the absorbance peak at 575 nm with little change in peak
position (Supporting Information, Figure S2). The Ca2þ
[Ca2þ
]free, where Amin is the absorbance of the free chela-
tor, Amax is the absorbance of the Ca2þ complex, and A is
the absorbance at an intermediate Ca2þ level, all measured
atthesamewavelength. The Hillplotfor absorbanceat320
nm gave a straight line with slope = 1 consistent with a 1:1
binding for compound 1 with Ca2þ. The x intercept
indicated the dissociation constant (Kd) of the complex
to be 509 nM. The binding characteristics of the closed
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Org. Lett., Vol. 13, No. 8, 2011

dissociation constant was found to be 164 μM from the
absorption studies, which agreed well within experimental
errorswiththatfrom emissiontitration. Complexation of2
with Mg2þ was also examined spectroscopically (Suppor-
ting Information, Figure S4). The Mg2þ dissociation con-
stant was found to be 14 mM from the fluorescence studies,
which is very similar as that for the closed form of com-
pound 1. Unlike Ca2þ chelation, the Mg2þ binding caused
little change in absorption spectra of 2. These results reflect
that Mg2þ binds primarily to only half of the chelator.
temperature in the dark. The above process can be re-
peated several cycles without significant changes in the
absorption spectrum. Addition of excess Ca2þ or Mg2þ to
the solution had little effect on the photoswitching, except
for the decrease of the absorbance at the photostationary
state probably due to the increased rate of thermal ring-
closure (Supporting Information, Figure S6ꢀ7). In the
presence of Ca2þ, the half-lifetime of the open form of 1
was decreased to 34 s. This value was found to be 28 s in the
presence of Mg2þ
.
In summary, a novel chelator 1 that reversibly binds and
releases Ca2þ in response to light was rationally designed
and synthesized to mimic calcium oscillations. A new
design concept was introduced for the development of
reversible Ca2þ chelators. Unlike previously reported
probes,4 in which the Ca2þ affinities were manipulated
through geometry changes or creation of additional bind-
ing sites, the Ca2þ affinity of 1 was modulated by harnes-
sing the changes in electronic properties of the BAPTA
moiety during photoswitching. Compound 1 exhibits high
affinity for Ca2þ (Kd: 509 nM) with excellent Ca2þ/Mg2þ
selectivity (Kd for Mg2þ: 19 mM) in the resting state. Upon
irradiation of UV light, compound 1 was converted to the
open isomer 1-open, which has a much lower affinity for
Ca2þ (Kd: 181 μM), while the affinity for Mg2þ is not
significantly affected (Kd: 14 mM). The open form reverts
back thermally, and the process can be repeated for several
cycles. To the best of our knowledge, this is the first demon-
stration of a reasonably satisfactory reversible Ca2þ che-
lator that might be used to generate defined waveforms of
calcium ions in a sample perhaps to mimic natural oscilla-
tions of calcium ions in cells and tissue, although increase in
the Ca2þ affinity of the resting state and further tuning of
the photochemical properties, i.e., the required wavelength
for photochemistry, thermal stability of the open isomer,
quantum yield of the photochemistry and optical control of
the ring-closure reaction, are still desired. Additional future
work in this direction is in progress.
Figure 4. Optical switching of 1 (20 μM, 10 mM MOPS, 100 mM
KCl, 1 mg/mL BSA, pH 7.2) with 312 nm UV light irradiation
for 30 s followed by thermal ring-closure in the dark at 20 °C. Inset
shows absorbance at 575 nm for five cycles of the switching process.
The optical switching properties of chelator 1 were
investigated in a buffered solution (10 mM MOPS, 100
mM KCl, pH 7.2). The formation of the open isomer 1-
open was not detected when a solution of 1 was irradiated
with UV light, probably because the lifetime of the open
state was too short to be detected.5 Bovine serum albumin
(BSA) was found to stabilize the open isomer probably by
interactions within the anion binding site of BSA. Thus,
BSA (1 mg/mL) was added to the solution in the following
studies. An example of photoswitching is shown in Figure
4. When 1 was irradiated with 312 nm UV light, a visible
absorption band (575 nm) corresponding to the open form
of 1 appeared. The photoinduced open form of 1 emitted at
around 600 nm. The absorption and emission spectra of 1-
open were very similar to these of compound 2, further
confirming that 2 is a good model compound for the open
form of 1. The open isomer converted back to the close
form thermally witha half-lifetime (t1/2) about 58 s at room
Acknowledgment. This work was financially supported
by NIH (PN2EY018241).
Supporting Information Available. Experimental pro-
cedures, characterization data for the new compounds,
and details of spectroscopic studies. This material is
available free of charge via the Internet at http://pubs.
acs.org.
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