Near-membrane iminocoumarin-based low affinity

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Near-membrane iminocoumarin-based low affinity
Cell Calcium (2002) 31(5), 221–227
© 2002 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0143-4160(02)00035-0, available online at http://www.idealibrary.com on
Near-membrane iminocoumarin-based
low affinity fluorescent Ca2+ indicators
F. Liepouri, 1 T. G. Deligeorgiev, 2 Z. Veneti, 3 C. Savakis, 3 H. E. Katerinopoulos 1
1
Department of Chemistry, University of Crete, Heraklion, 71 409 Crete, Greece
Faculty of Chemistry, University of Sofia, 1126 Sofia, Bulgaria
3
School of Medicine, University of Crete, Heraklion, 71 409 Crete, Greece
2
Summary Two new potential near-membrane iminocoumarin-based fluorescent Ca2+ indicators were synthesized and
the spectral profiles of their free and Ca2+ bound forms were studied. The probes incorporate in their BAPTA-related
structures, the 3-(benzimidazolyl)iminocoumarin or the 3-(benzothiazolyl)iminocoumarin moiety, substituted at the imino
nitrogen with an n-dodecyl lipophilic chain. The compounds are excited with visible light and have Ca2+ dissociation
constant values of 5.50 and 4.49 ␮M, respectively, the highest reported to date in the literature. Fluorescence spectra
studies indicated a clear shift in their excitation wavelength maxima upon Ca2+ binding along with changes in fluorescence
intensity that enable the compounds to be used as ratiometric near-membrane, low Ca2+ affinity probes. © 2002 Elsevier
Science Ltd. All rights reserved.
INTRODUCTION
Changes in intracellular calcium ion concentrations
([Ca2+ ]i ) are frequently the result of Ca2+ transport into
the cell via membrane calcium channels and transporters
[1,2]. As a result, [Ca2+ ]i at the inner-membrane vicinity
may, at least transiently, reach levels significantly different from those in the cytosol. The synthesis of lipophilic
fluorescent probes that can be introduced into the cell, associate to the inner-membrane surface and selectively respond to [Ca2+ ]i changes only in their immediate vicinity,
provided a valuable tool for detection of these localized
calcium concentration changes.
The first lipophilic probe reported was C18-Fura-2, a
compound based on the structure of the popular calcium
indicator Fura-2 [3] with an extension of an octadecane
chain linked at the 4-position of its aminophenol moiety
via a thiourea-linker [4]. Fluorescence imaging studies indicated that the probe was linked to the cell membrane.
Cell depolarization experiments using C18-Fura-2 and
Fura-2 simultaneously showed a distinct difference in the
rate of increase of [Ca2+ ]i at the cell membrane vicinity
and the cytosol. Disadvantages in the use of C18-Fura-2
include (a) its high affinity for Ca2+ (K d = 150 nM), (b) the
inability of its pentaacetoxymethyl ester to load into cells
Correspondence to: Haralambos E. Katerinopoulos, Department of Chemistry,
University of Crete, Heraklion, 71 409 Crete, Greece. Tel.: +30-810-393626;
fax: +30-810-393601; e-mail: [email protected]
due to its high lipophilicity that prevents the molecule
from reaching the intracellular esterases, and (c) the dependence of its fluorescence on the concentration of the
Ca2+ -dye complex.
A modification of the side-chain in C18-Fura-2 yielded
FFP18. In this molecule an N-n-dodecylpiperazine moiety was connected to the aromatic ring through a
propionyl-linker [5]. Protonation of the piperazine nitrogen at physiological pH values significantly reduced
leakage of the molecule through the cell membrane and
increased its Kd as shown by fluorescence imaging and
Ca2+ titration experiments. Comparative membrane depolarization experiments [6] with FFP18 and Fura-2 indicated differences in the kinetics of the two probes: FFP18
reached maximum fluorescence in 12 ms whereas Fura-2
responded in 150 ms. Maximum Ca2+ concentrations
detected by FFP18 were 1 mM, twice as high as those detected by Fura-2. FFP18 has a higher calcium dissociation
constant value than C18-Fura-2 (K d = 510 nM) and has
the relative advantage of being cell membrane permeable,
but only at a 50% fraction. It also has the disadvantage
that its fluorescence is dependent on the concentration
of the Ca2+ -dye complex. FIP18 was designed after FFP18
with its side-chain incorporated in the Indo-1 framework;
its Kd value is 400 nM [7]. The spectral profiles of the aforementioned UV excitable dyes are very similar to those
of their parent molecules. Calcium Green-C18 [8] and
Fura Indoline-C18 [9] are excitable by visible light dyes
221
222
F Liepouri, TG Deligeorgiev, Z Veneti, C Savakis, HE Katerinopoulos
with respective Kd values of 280 and 260 nM, their structures being based on the framework of Calcium Green
and Fura-2, respectively. It is apparent that all of these
mentioned dyes would be saturated, should the transient near-membrane concentrations reach micromolar
levels.
In this report, we describe the synthesis and the spectral profile studies of C12-BIIC and C12-BTIC, two new
iminocoumarin-based low affinity calcium indicators. The
choice of the chromophore was based on two facts: (a) the
ability of the iminocoumarin moiety to be extended via
nitrogen substitution with a lipophilic chain, and (b) the
structural similarity of the new probe with BTC, a low affinity calcium indicator [10,11], that could guarantee a similar Ca2+ binding profile for the potential near-membrane
probes.
MATERIALS AND METHODS
Melting points were taken on a Thomas–Hoover apparatus and are uncorrected. NMR spectra were taken on
an AMX500 Bruker FT-NMR spectrometer; proton chemical shifts are reported in relative to tetramethylsilane.
Mass spectra were recorded on a Shimatzu ESI-MS mass
spectrometer. Fluorescence spectra were recorded on an
Aminco Bowman spectrofluorimeter (Spectronics Instruments, Inc., New York, USA). Standard free Ca2+ concentration buffers were purchased from Molecular Probes,
Inc. (Eugene, OR, USA, Calcium Calibration Buffer Kit #3,
C-6775).
(6 mg, 0.133 mmol). The system was stirred at room temperature for 1 h. The organic solvent was removed in
vacuo and the pH of the aqueous solution was adjusted
to 7.0 by addition of traces of 1N HCl solution. The solvent was removed in vacuo and the solid residue was
purified by repeated (10 times) suspension in acetone
and centrifugation and repeated (five times) suspension
in acetonitrile and centrifugation. The yellow solid was
dried under vacuum. The product yield was 8 mg (86%).
1
H NMR (D2 O, 500 MHz): 0.7–1.33 (m, 21H), 2.16–2.33
(m, 5H), 3.52 (m, 2H), 3.76 (s, 4H), 4.01 (s, 4H), 4.29 (s, 4H),
6.53 (s, 1H), 6.70 (br.s, 3H), 6.92 (s, 1H), 7.30 (s, 2H), 7.63
(br.s, 2H), 8.68 (s, 1H). ESI-MS (negative ionization) m/z:
841 (M − 1)− .
Synthesis of C12-BTIC tetramethyl ester
In a flame dried round-bottomed flask equipped with
a magnetic stirrer were placed iminocoumarin 2 [12]
(122 mg, 0.16 mmol), dodecylamine hydrochloride (37 mg,
0.17 mmol) and 10 ml of dry ethanol. The system was
Synthesis of C12-BIIC tetramethyl ester
In a flame dried round-bottomed flask equipped with a
magnetic stirrer and condenser were placed iminocoumarin 1 [12] (100 mg, 0.137 mmol), dodecylamine hydrochloride (30 mg, 0.137 mmol) and 2 ml of dry ethanol.
The system was heated under reflux for 5 h. The solvent
was removed and the product was purified with flash
chromatography (Merck silica gel 60, mesh 230–400) using 30% ethanol in petroleum ether as eluent. The product
yield was 120 mg (94%). 1 H NMR (CDCl3 , 500 MHz): 0.90
(t, J1 = 6.6 Hz, 3H), 1.2–1.6 (m, 18H), 1.79–1.84 (m, 2H),
2.30 (s, 3H), 3.63 (s, 6H), 3.64 (s, 6H), 4.16 (s, 4H), 4.27
(s, 4H), 4.30 (br.s, 4H), 6.61 (s, 1H), 6.70 (s, 1H), 6.72 (d,
J = 8.7 Hz, 1H), 6.79 (d, J = 8.0 Hz, 1H), 6.92 (s, 1H), 7.52
(d, J = 7.0 Hz, 1H), 7.79 (d, J = 7.2 Hz, 1H), 8.50 (s, 1H).
MS-APCI (positive ionization) m/z: 898 (M + 1)+ , 920
(M + Na)+ .
Synthesis of C12-BIIC tetralithium salt
In a round-bottomed flask were placed tetramethyl ester
3 (10 mg, 0.011 mmol), 1 ml THF, 1 ml H2 O, and LiOH
Cell Calcium (2002) 31(5), 221–227
Scheme 1 Preparation of fluorescent calcium probes C12-BIIC
and C12-BTIC.
© 2002 Elsevier Science Ltd. All rights reserved.
Iminocoumarin-based low affinity fluorescent Ca2+ indicators
heated at 80 ◦ C for 10 h. The product was filtered, washed
with cold methanol and purified with flash chromatography (Merck silica gel 60, mesh 230–400) using 10–20%
ethyl acetate in petroleum ether as eluent. The product
yield was 98 mg (67%). 1 H NMR (CDCl3 , 500 MHz): 0.90
(t, J = 7.0 Hz, 3H), 1.2–1.6 (m, 18H), 1.83 (t, J = 6.5 Hz,
2H), 2.30 (s, 3H), 3.64 (s, 6H), 3.65 (s, 6H), 4.17 (s, 4H), 4.27
(s, 4H), 4.31 (br.s, 4H), 6.58 (s, 1H), 6.70 (s, 1H), 6.72 (d,
J = 8.0 Hz, 1H), 6.79 (d, J = 8.0 Hz, 1H), 6.97 (s, 1H), 7.38 (t,
J = 7.2 Hz, 1H), 7.50 (d, J = 7.2 Hz, 1H), 7.96 (d, J = 7.8 Hz,
1H), 8.0 (d, J = 7.8 Hz, 1H), 8.45 (s, 1H). MS-APCI (positive
ionization) m/z: 915 (M + 1)+ .
Synthesis of C12-BTIC tetralithium salt
In a round-bottomed flask were placed tetramethyl ester 4
(10 mg, 0.011 mmol), 1 ml THF, 1 ml H2 O, and LiOH (6 mg,
0.133 mmol). The system was stirred at room temperature
for 1 h. The organic solvent was removed in vacuo and the
pH of the aqueous solution was adjusted to 7.0 by addition
of traces of 1N HCl solution. The solvent was removed
in vacuo and the solid residue was purified by repeated
(five times) suspension in acetone and centrifugation. The
yellow solid was dried under vacuum. The product yield
was 8 mg (85%). 1 H NMR (D2 O, 500 MHz): 0.70–1.33 (m,
21H), 2.18 (br.s, 3H), 3.47–3.59 (m, 6H), 3.75–4.50 (m, 8H),
6.40–6.50 (m, 1H), 6.70 (m, 2H), 6.80–6.95 (m, 3H), 7.16
(br.s, 1H), 7.30–7.50 (m, 1H), 7.55–7.78 (m, 1H), 7.78–7.95
(m, 1H) ESI-MS (negative ionization) m/z: 857 (M − 1)− .
223
Preparation of indicator solutions containing
adjusted Ca2+ concentrations
Stock solutions of the Ca2+ indicators (salt form) were prepared in nanopure water at 0.2 mM concentration. A 10 ␮l
aliquot of each stock solution was then added to 2 ml samples of commercially available calcium calibration buffers
to make a final indicator concentration of 1 ␮M. Calcium
Calibration Buffer Kit #3 contains 10 ml of zero-free Ca2+
buffer (10 mM K2 EGTA) and 10 ml each of 10 solutions
with free Ca2+ concentrations ranging from 1 ␮M to 1 mM.
The K2 EGTA/CaEGTA buffering system is employed to provide the two lowest concentrations (1.35 and 2.85 ␮M). At
higher concentrations (5 ␮M and above), the Ca2+ level
is not buffered and is verified using an ion-selective electrode. All solutions in the kit contain 100 mM KCl and
10 mM MOPS, pH 7.2, and are prepared in deionized water
(resistance ≥ 17.9 M) [11].
Fluorescence microscopy experiments
Drosophila melanogaster embryos were placed on double
stick tapes fixed on glass slides, desiccated in room air
and microinjected under Halocarbon 700 oil (Sigma) with
50 ␮M solutions of BIIC, BTIC, C12-BIIC, and C12-BTIC
in nanopure water. Microinjections were performed using glass needles with 2–5 ␮m bore and a manual Narishige IM 300 Microinjector (Narishige Medical Systems,
Greenveal, NY, USA). Specimens were immediately transferred to the confocal microscopy facility where optical
Fig. 1 Excitation spectra of 1 ␮M C12-BTIC tetralithium salt as a function of increasing Ca2+ concentration in free Ca2+ concentration
buffers: (a) 0 ␮M, (b) 1.35 ␮M, (c) 2.85 ␮M, (d) 10.0 ␮M, (e) 20.0 ␮M, (f) 30.0 ␮M. Spectra were obtained at 22 ◦ C in an Aminco Bowman
Series 2 fluorimeter with excitation and emission slit widths set at 4 nm. The solutions were scanned from 350 to 530 nm with the emission
collected at 537 nm.
© 2002 Elsevier Science Ltd. All rights reserved.
Cell Calcium (2002) 31(5), 221–227
224
F Liepouri, TG Deligeorgiev, Z Veneti, C Savakis, HE Katerinopoulos
Fig. 2 Emission spectra of 1 ␮M C12-BTIC tetralithium as a function of increasing Ca2+ concentration in free Ca2+ concentration buffers: (a)
0 ␮M, (b) 1.35 ␮M, (c) 2.85 ␮M, (d) 10.0 ␮M, (e) 20.0 ␮M, (f) 30.0 ␮M. Spectra were obtained at 22 ◦ C in an Aminco Bowman Series 2
fluorimeter with excitation and emission slit widths set at 4 nm. The excitation wavelength was 421 nm.
sections were made using a confocal laser scanning microscope Leica TCS-NT using the 10× objective. Z-series
of optical sections were obtained in every case and were
projected along the z-axis to obtain a general view of the
Drosophila embryos. The excitation wavelength was set at
476 nm.
RESULTS AND DISCUSSION
Chemistry
The synthesis of C12-BIIC and C12-BTIC is depicted
in Scheme 1. The reaction of iminocoumarins 1 and 2
[12] with dodecylamine hydrochloride involves initial
Fig. 3 Determination of C12-BTIC dissociation constant for Ca2+ . The Kd was calculated from excitation intensity values, according to
Tsien’s algorithm for ratiometric dyes [3].
Cell Calcium (2002) 31(5), 221–227
© 2002 Elsevier Science Ltd. All rights reserved.
Iminocoumarin-based low affinity fluorescent Ca2+ indicators
protonation of the iminocoumarin nitrogen followed by
attack of dodecylamine to the charged intermediate and
subsequent formation of the dodecylimines 3 and 4 (C12BIIC and C12-BTIC tetramethyl esters, respectively) by
225
acid catalyzed cleavage of the amine group [13]. The derivatized iminocoumarins are no longer sensitive to aqueous
treatment allowing for basic (LiOH, 12 eq./THF/H2 O) hydrolysis of the methyl esters to yield 5 and 6 (C12-BIIC
Fig. 4 Confocal fluorescence microscopy projections of Drosophila melanogaster embryos microinjected with 50 ␮M solutions of the dyes in
nanopure water. Cytosol Ca2+ indicators BIIC and BTIC are homogeneously distributed throughout the cell (A and B) whereas potential
near-membrane indicators C12-BIIC and C12-BTIC are located at the inner-membrane region (C and D).
© 2002 Elsevier Science Ltd. All rights reserved.
Cell Calcium (2002) 31(5), 221–227
226
F Liepouri, TG Deligeorgiev, Z Veneti, C Savakis, HE Katerinopoulos
Fig. 4 (Continued ).
and C12-BTIC tetralithium salts). All compounds were
characterized by NMR and mass spectroscopy.
Fluorescence spectral properties
The excitation spectrum of C12-BIIC exhibited a wavelength maximum at 469 nm, which shifts to 401 nm upon
calcium binding. A small but distinct shift is also observed
at the emission maxima of the free (515 nm) and the
Ca2+ bound form (498 nm) of the probe. The fluorescence
signal intensity increased by 585% upon calcium binding at saturation Ca2+ levels. The excitation maximum
of the free form of C12-BTIC, the benzothiazolyl substituted analog, is at 471 nm whereas its bound form has a
wavelength maximum at 421 nm as shown in Fig. 1. This
compound exhibits a 537 nm emission maximum in both
forms (Fig. 2). The fluorescence signal intensity in this case
increased by 181% upon calcium binding at saturation
Ca2+ levels. The extinction coefficients of the absorption
spectra of the free and calcium bound forms of C12-BIIC
are 2.2 × 104 and 2.0 × 104 M−1 cm−1 and the respective
values for C12-BTIC are 1.7 × 104 and 0.9 × 104 M−1 cm−1 .
These values are comparable to those of C18-Fura-2
(2.5 × 10−4 and 3.0 × 10−4 M−1 cm−1 ), however, in the latter dye the value of ε increases upon calcium binding;
such a change in ε is also observed for FFP18.
The absence of an isosbestic point at higher [Ca2+ ], a
phenomenon also observed in the excitation spectra of
C18-Fura-2 [4] and FFP18 [6], could tentatively be explained by similar arguments to those expressed by Etter
et al. [4]. In an aqueous environment and at high dye concentrations, the lipophilic chains might cluster, bringing
the fluorophores close enough to interact. At lower [Ca2+ ]
Cell Calcium (2002) 31(5), 221–227
the majority of molecules maintain a total charge of +4,
repelling each other, and thus, keeping the chromophore
moieties apart. However, at near saturating [Ca2+ ], this
charge is partially neutralized by the presence of the calcium ion, allowing the chromophores to reach each other
and probably form complexes with altered electronic
structure.
Ca2+ binding constants
The dissociation constant (Kd ) value of C12-BIIC, calculated according to Tsien’s algorithm for ratiometric
dyes [3], is 5.50 ␮M, indicating that this probe has the
lowest affinity of all near-membrane probes reported to
date. Compared to Fura- and Indo-type near-membrane
indicators, FFP18 and FIP18, which also carry a dodecyl
lipophilic chain, C12-BIIC has an 11- and 14-times lower
affinity, respectively. A similar (4.49 ␮M) Kd value was
calculated for C12-BTIC (Fig. 3). This probe has a 9- and
11-times lower affinity for Ca2+ , compared to those of
FFP18 and FIP18, respectively.
Fluorescence microscopy experiments
The potential utility of C12-BIIC and C12-BTIC as
near-membrane indicators was shown by preliminary
fluorescence microscopy experiments in single cells. Confocal fluorescence microscopy projections of Drosophila
melanogaster embryos, microinjected with 50 ␮M solutions of the dyes in nanopure water, indicated a clear
difference of spatial distribution. Cytosol Ca2+ indicators
BIIC (1) and BTIC (2) [12] are homogeneously distributed
throughout the cell (Fig. 4A and B), whereas potential
© 2002 Elsevier Science Ltd. All rights reserved.
Iminocoumarin-based low affinity fluorescent Ca2+ indicators
near-membrane indicators C12-BIIC and C12-BTIC are
located at the inner-membrane region (Fig. 4C and D).
CONCLUSION
Two new potential near-membrane Ca2+ indicators have
been synthesized and their fluorescence spectra profiles,
in the presence of increasing Ca2+ concentrations, were
studied. The structure of these compounds is based on
the BAPTA framework incorporating the N-n-dodecyl-3(benzimidazolyl)iminocoumarin and N-n-dodecyl-3-(benzothiazolyl)iminocoumarin chromophores. The n-dodecyl
substituent was added with the purpose of enabling the
compounds to be used as near-membrane indicators.
Fluorescence spectra studies indicated that the probes exhibit a clear shift in excitation wavelength maxima upon
Ca2+ binding and, therefore, can be used as ratiometric calcium indicators. The dissociation constant of both
probes is in the micromolar range. Their Kd values of 5.50
and 4.49 ␮M, the highest reported to date, are indicative
of the potential utility of the probes as [Ca2+ ]i indicators
at the inner-membrane vicinity where transient calcium
concentrations may reach levels that are saturating the
near-membrane indicators currently used. The use of such
low affinity indicators has been suggested by Etter et al.
[4] as well as by Vergara and his collaborators [14] who
mentioned advantages in the use of lower affinity dyes
responding to high Ca2+ spikes generated by laser flash
photolysis of DM-nitrophen. In recent studies, Neher and
coworkers [15] concluded that “synaptic depression at the
calyx of Held is governed by localized, near-membrane
[Ca2+ ]i signals not visible to the indicator dye (Fura-2) or
else by an altogether different mechanism” and also indicated that “the limited spatial and temporal resolution of
ratiometric methods still prevents direct measurement of
the submembrane [Ca2+ ]i at the sites of secretion” of catecholamines from neuroendocrine cells [16]. Employment
of the near-membrane indicators in these as well as in
other [17] related studies might be a useful contribution
in unraveling the role of calcium in the function of such
biologically important systems.
ACKNOWLEDGEMENTS
Grant No. 1224 of the Special Research Account of the
University of Crete, an EPEAEK Grant by the EU and the
Greek Ministry of Education, and a Greek–Bulgarian Educational Exchange Grant by the Greek Ministry of Education have supported this work. The authors would like to
express their gratitude to Dr. E. Foukaraki for useful discussions during the project and I. Livadaras for excellent
technical assistance.
© 2002 Elsevier Science Ltd. All rights reserved.
227
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