Synthesis and characterization of a new red-emitting Ca2+ indicator, calcium ruby

Article abstract of DOI:10.1021/ol070648h

Equation Presented Calcium Ruby m-Cl (X = H, Y = Cl) is a visible-light excited red-emitting calcium concentration ([Ca²⁺]) indicator dye (579/598 nm peak excitation/emission) with a side arm for conjugation via EDC or click chemistry. Its larg

Full text of DOI:10.1021/ol070648h

ORGANIC
LETTERS
2007
Vol. 9, No. 14
2629-2632
Synthesis and Characterization of a New
+
Red-Emitting Ca2 Indicator, Calcium
Ruby
Ste´phane Gaillard,† Aleksey Yakovlev,‡,§ Camilla Luccardini,‡,§ Martin Oheim,§
Anne Feltz,‡ and Jean-Maurice Mallet*,†
De´partement de Chimie, Ecole Normale Supe´rieure, ENS-CNRS UMR 8642,
24 rue Lhomond, Paris, F-75005 France, Laboratoire de Neurobiologie,
De´partement de Biologie, Ecole Normale Supe´rieure, ENS-CNRS UMR 8544,
46 rue d’Ulm, Paris, F-75005 France, and Laboratory of Neurophysiology and New
Microscopies, UniVersity Paris Descartes, INSERM, U603, 45 rue des Saints Pe`res,
Paris, F-75006 France
jean-maurice.mallet@ens.fr
Received March 26, 2007
ABSTRACT
+
Calcium Ruby m-Cl (X
) H, Y )
Cl) is a visible-light excited red-emitting calcium concentration ([Ca2 ]) indicator dye (579/598 nm peak
excitation/emission) with a side arm for conjugation via EDC or click chemistry. Its large molar extinction and high quantum yield rank it
among the brightest long-wavelength Ca2 indicators. Calcium Ruby is a promising alternative to existing dyes for imaging [Ca2 ] in multicolor
+
+
fluorescence applications or in the presence of yellow-green cellular autofluorescence.
Measuring the temporal and/or spatial variations in the
intracellular free calcium concentration ([Ca2+]i) is a common
theme in cytometry, cell biology, and neuroscience. Follow-
ing the seminal work by Tsien and co-workers in the early
1980s,1,2 a whole family of fluorescent indicators with
different fluorescence properties and Ca2+-binding affinities
has been generated.3,4 These indicators have in common the
principle of covalently linking an organic fluorophore to a
Ca2+-chelating moiety, mostly to BAPTA. Ca2+ binding
rotates the carbon-nitrogen bond which diverts the nitrogen
free electron pairs out of conjugation, resulting in modulating
the fluorescence intensity and/or in spectral shifts. Most of
these Ca2+ indicators and certainly the most commonly used
are fluorescein-derived fluorophores and hence emit green
to yellow fluorescence upon blue-turquoise excitation.
† De´partement de Chimie, Ecole Normale Supe´rieure.
‡ De´partement de Biologie, Ecole Normale Supe´rieure.
§ University Paris Descartes.
However, for many cell and neurobiological applications,
red-emitting Ca2+-sensitive dyes would offer a distinctive
advantage over green-emitting probes. Visible-light excited
red emission would permit a better discrimination against
cellular autofluorescence, allow the detection of Ca2+ release
generated by photoreceptors,5 and be compatible with the
use of photoactivated “caged” Ca2+ compounds. Most
(1) Tsien, R. Y. Biochemistry 1980, 19, 2396-2404.
(2) Grynkiewicz, G.; Poenie, M.; Tsien, R. Y. J. Biol. Chem. 1985, 260,
3440-3450.
(3) Takahashi, A.; Camacho, P.; Lechleiter, J. D.; Herman, B. Physiol.
ReV. 1999, 79, 1089-1125.
(4) Katerinopoulos, H. E.; Foukaraki, E. Curr. Med. Chem. 2002, 9, 275-
306.
10.1021/ol070648h CCC: $37.00
© 2007 American Chemical Society
Published on Web 06/07/2007

Table 1. Comparison of the Spectral Properties of Calcium Ruby-Cl with Other Red-Emitting Ca2+-Sensitive Dyesa
λmabasx/λmax
(nm)
ꢀ
em
b
(M-1 cm-1
)
æCa -free/æCa
Fmax/Fmin
2+
2+-bound
Calcium Orange
Rhod-118
Rhod-2
X-Rhod-1
ICPBC20
549/576
556/578
553/576
574/594
(558)485/(586)582d
579/598
590/615
80000
e
82000
92000
e
e
∼3
0.0014/0.021
0.03/0.102
e
e
0.026/0.42
e
e
14 in situ,3 100c,10
4-8 in situ,19 100c,10
0.25
32
∼2.55
Calcium Ruby
Calcium Crimson
100000
113,000
a ꢀ ) molar extinction, æ ) quantum yield, F ) fluorescence at peak emission. b Defined as the relative change in fluorescence at peak emission wavelength.
c Depending on the purification (no data available). d (Ca2+-free) Ca2+-bound. e No data available.
important, a red Ca2+ sensor would also permit the simul-
taneous detection of [Ca2+]i and enhanced green fluorescent
protein (EGFP) in a dual-emission detection scheme. The
green-yellowish emission of EGFP results in an appreciable
cross-talk that obscures the fluorescence emission of most
current Ca2+ indicators.6
was considered to be less interfering with the functional part
of the indicator and more versatile than the aromatic ring
anchoring used earlier by Gee and co-workers in Fluo-4 con-
jugates.7 The linker offers a terminal azido group for conju-
gation, either via click chemistry using acetylenic-tagged
molecules or through EDC-assisted coupling to a COOH bear-
ing carrier after reduction to an amino group. These reactions
permit the attachment of Calcium Ruby to a dextran, such
as for retrograde labeling,8 or its attachment to the surface
of colloidal semiconductor nanocrystals in a hybrid bioassay
combining inorganic and organic fluorophores.9
Advantageously, substitutions on the BAPTA moiety
enable us to precisely control the indicator apparent Ca2+-
binding affinity, Kd(Ca2+), denoted Kd in the sequel. By
substituting the BAPTA in positions X and/or Y, we generate
a whole family of red-emitting Ca2+ dyes with Kd values in
the 0.5-300 µM range. In the present work, we specifically
describe the chloride-substituted Calcium Ruby.
The plot of the Hammett constants σ of the substitutions
on the aromatic ring of the BAPTA system of known
BAPTA-based Ca2+ dyes against their log(Kd) (dissociation
constant) displays a good correlation (Figure 2). From this
relationship, we would anticipate a Kd on the order of 20-
25 µM for the chloride-substituted Calcium Ruby molecule
(σ ) 0.37). Such a low-affinity Ca2+ indicator is of particular
use when imaging Ca2+ microdomains, that is, highly
localized and fast, transient micromolar excursions from
resting [Ca2+]i that are observed, for example, in response
to voltage-gated Ca2+ channel opening or localized release
from internal Ca2+ stores.12,13
Among the long-wavelength Ca2+ indicators available, the
X-Rhod family and Calcium Crimson result from an expan-
sion of the rhodamine chromophore into a seven-ring system
of a Texas Red-type fluorophore. Despite their red-shifted
emission, the rhodamine derivatives Calcium Orange (549/
575 nm peak absorbance/peak emission), Rhod-2 (553/576
nm), X-Rhod-1 (574/594 nm), and Calcium Crimson (590/
615 nm), see Table 1, are not being widely used, either due
to their intracellular compartmentalization, small absorption
cross-section, inappropriate Kd value, or because they are
simply no longer commercialized. Hence, the demand for
red-emitting Ca2+ indicators displaying little spectral overlap
with EGFP and cellular autofluorescence is still strong.
In this letter, we describe the synthesis of a visible-light
excited red-emitting Ca2+ dye with a BAPTA moiety grafted
on an extended rhodamine fluorophore (Figure 1). The novel
Synthesis. The synthesis of the BAPTA part of 11,
depicted in Scheme 1, started from the commercially
(7) Martin, V. V.; Beierlein, M.; Morgan, J. L.; Rothe, A.; Gee, K. R.
Cell Calcium 2004, 36, 509-514.
(8) Beierlein, M.; Gee, K. R.; Martin, V. V.; Regehr, W. G. J.
Neurophysiol. 2004, 92, 591-599.
Figure 1. Calcium Ruby-Cl, compound (11) in Scheme 1.
(9) Snee, P. T.; Somers, R. C.; Nair, G.; Zimmer, J. P.; Bawendi, M.
G.; Nocera, D. G. J. Am. Chem. Soc. 2006, 128, 13320-13321.
(10) Haugland, R. P. The Handbook: A Guide to Fluorescent Probes
and Labeling Technologies, 10th ed.; Invitrogen: 2006; p 1126.
(11) Physical Organic Chemistry; Hine, J., Ed.; McGraw-Hill: New
York, 1962; Chapter 4, p 81.
Calcium Ruby indicator also includes a linker side chain that
we attached to the ethylene glycol bridge. This bridge anchor
(12) Bootman, M. D.; Lipp, P.; Berridge, M. J. J. Cell Sci. 2001, 114,
2213-2222.
(13) Oheim, M.; Kirchhoff, F.; Stu¨hmer, W. Cell Calcium 2006, 40, 423-
439.
(5) Ukhanov, K.; Payne, R. J. Neurosci. 1997, 17, 1701-1709.
(6) Bolsover, S.; Ibrahim, O.; O’luanaigh, N.; Williams, H.; Cockcroft,
S. Biochem. J. 2001, 356, 345-352.
2630
Org. Lett., Vol. 9, No. 14, 2007

Scheme 1. Synthesis of 11
8-hydroxy julolidine in hot and acid conditions. In our hands,
the drastic conditions needed for this reaction were not
successful but rather led to the formation of an intractable
mixture of colored products. Thus, we preferred to preform
the diphenyl ether part 10. The known diamino diphenyl
ether14 is octa-alkylated15 with 1-bromo 3-chloropropan to
give 10 (Scheme 1).
The coupling reaction between 8 and 10 was conducted
in neat trifluoroacetic acid (TFA) at room temperature.16,17
The intermediate was oxidized by oxygen bubbling in
dichloromethane, and the four ester functions were saponified
using methanolic KOH. The final product was purified on
TLC plate RP18 using MeOH/AcOH 70:3.
Absorption. As expected, the absorbance spectrum of
Calcium Ruby-Cl (11) was similar to that of other dyes of
the X-Rhod family, with a 2-3 nm shift to longer wave-
lengths. Absorbance was maximal at 578-580 nm (n ) 4)
and did not appreciably change upon Ca2+ binding (between
nominally 0-1.2 mM [Ca2+], data not shown).
Due to the hygroscopic nature of the product, we estimated
the concentrations of 11 solutions using a extinction coef-
ficient ꢀ of ∼100 000 M-1 cm-1, as reported for other
X-Rhod dyes. We next determined the [Ca2+] dependence
of the fluorescence of 11 using the titration technique of
Minta et al.18
Figure 2. Correlation between log(Kd) (from ref 10) versus
Hammett constants11 for different aromatic substitutes of BAPTA
at position X and Y (2, Fluo; O, Rhod; 0, X-Rhod series of Ca2+
sensitive dyes; and [, Calcium Ruby-Cl).
-
available 1,2-epoxy-7-octen. The epoxy ring was opened with
potassium o-nitrophenate to give mainly the secondary
alcohol 1. This alcohol was mesylated, and the mesylate 2
reacted with potassium nitrochlorophenate to give 3. Reduc-
tion of the two nitro groups and alkylation of the resulting
amino groups with methyl bromoacetate in the presence of
diisopropyl ethyl amine (DIEA) gave the key intermediate
5. Vilsmeier formylation of 5 was followed by the double
bond activation. This was achieved in a two-step proce-
dure: ionic addition of HBr in dichloromethane (DCM) then
substitution of the bromo to an azido group using NaN3 in
DMF.
(14) Critchley, J. P.; Grattan, P. A.; White, M. A.; Pippet, J. S. J. Polym.
Sci. 1972, 10, 1789-1807.
(15) Kauffman, J. M.; Imbesi, S. J.; Abdul-Aziz, M. Org. Prep. Proc.
Int. 2001, 33, 603-613.
(16) de Silva, A. P.; Gunaratne, H. Q. N.; Kane, A. T. M.; Maguire, G.
E. M. Chem. Lett. 1995, 125-126.
In classical syntheses of X-Rhod dyes, the aromatic
aldehyde (such as 8) is opposed to the commercially available
(17) Woods, L. L.; Sapp, J. J. Org. Chem. 1962, 27, 3703-3705.
Org. Lett., Vol. 9, No. 14, 2007
2631

Fluorescence. 11 exhibits a fluorescence emission spec-
trum similar to that of other X-Rhod derivatives (see ref 4
for review) with a peak at 598-600 nm. Peak fluorescence
increased ∼32-fold upon Ca2+ binding, with no appreciable
spectral shift (Figure 3). As with other rhodamine derivatives,
comparable to that of X-Rhod-1 and Rhod-2 fluorophores
and a high dynamic range upon Ca2+ binding relative to other
red-emitting fluorophores; see Table 1 for a quantitative
comparison.
Ca2+-Binding Affinity. Calcium Ruby-Cl with a chloride
in the meta position (Figure 1) had an apparent dissociation
constant (30.3 ( 2.6 µM, n ) 4), measured at 20 °C and
0.1 M ionic strength. This value is in the range predicted
for a chloride-substituted BAPTA (Figure 2).
In summary, our results indicate that we have successfully
synthesized a visible-light red-emitting [Ca2+] indicator dye
(579/598 nm peak excitation/emission). Its large molar
extinction and high quantum yield (ꢀæ ∼ 42 000 M-1 cm-1)
ranks our novel Calcium Ruby Ca2+ indicator among the
brightest red-fluorescent Ca2+-sensitive dyes. Its high pho-
tostability and large red shift compared to existing Ca2+-
sensitive dyes should make the Calcium Ruby family a
versatile alternative to conventional green-yellow and orange-
emitting organic Ca2+ indicators. With its spectral properties,
they respond to the demands of many biological multicolor
fluorescence applications, specifically when imaging cells
expressing enhanced green fluorescent protein (EGFP).
Figure 3. Fluorescence emission upon 535 nm excitation of 11
with [Ca2+] ranging from 0.21 (bottom trace) to 329 µM (top).
Titration buffer contained 100 mM KCl, 30 mM MOPS (pH 7.2),
10 mM NTA, and 0.01 mM Calcium Ruby-Cl.
Acknowledgment. The authors would like to thank J.
Neyton (De´partement de Biologie, Ecole Normale Su-
pe´rieure) for discussion, N. Ropert and S. Charpak (Uni-
versity Paris Descartes) for comments on earlier versions
on the manuscript. We thank L. Jullien (ENS) for access to
the ENS spectrometric platform. Funded by the European
Union (STRP 013880 “Long-Term Observation of Single
Biomolecules by Novel Minimal Invasive Fluorescence
Lifetime Imaging Nanoscopy”), the French Ministry of
Research and Technology (ACI nanosciences), the Agence
Nationale de la Recherche (ANR PNANO). A.Y. and C.L.
were recipients of CNRS and EU postdoctoral fellowships.
S.G. acknowledges a postdoctoral fellowship from the ANR.
fluorescence was very photostable, particularly when com-
pared to that of fluorescein (not shown).
Quantum Yield æ. Comparison with Sulforhodamine 101
and Rhodamine B yielded an estimate of æ ) 0.42 ( 0.03
(n ) 4) for the quantum yield of 11 in the presence of 329
µM free Ca2+. Ca2+ binding increased the quantum yield
∼17-fold compared to that of the Ca2+-free form (0.026 (
0.001, n ) 4). In summary, 11 has a low basal fluorescence
Supporting Information Available: Additional experi-
mental details. This material is available free of charge via
the Internet at http://pubs.acs.org.
(18) Minta, A.; Kao, P. Y.; Tsien, R. Y. J. Biol. Chem. 1989, 264, 8171-
8178.
(19) Gerenceser, A. A.; Adam-Vizi, V. Biophys. J. 2005, 88, 698-714.
(20) Roussakis, E.; Liepouri, F.; Nifli, A. P.; Castanas, E.; Deligeorgiev,
T. G.; Katerinopoulos, H. E. Cell Calcium 2006, 39, 3-11.
OL070648H
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Org. Lett., Vol. 9, No. 14, 2007