An efficient fluorescent probe for ratiometric pH measurements in aqueous solutions

Article abstract of DOI:10.1002/anie.200460557

Just as litmus paper changes from red to blue with an increase in pH, so the fluorescence emission of the oxazole derivative 1 shifts from green to blue. Ratiometric measurements of the fluorescence emissions of the two species, after one- or two-photon e

Full text of DOI:10.1002/anie.200460557

Angewandte
Chemie
pH Sensors
An Efficient Fluorescent Probe for Ratiometric
pH Measurements in Aqueous Solutions
Sandrine Charier, Odile Ruel, Jean-Bernard Baudin,
Damien Alcor, Jean-François Allemand, Adrien Meglio,
and Ludovic Jullien*
The development of new powerful microscopy approaches,[1–4]
as well as the availability of various synthetic and natural
fluorescent molecules,[5–7] has made fluorescence microscopy
an essential tool for cellular biology. Fluorescent labels that
specifically target molecules or organelles are currently used
to investigate the structures and functions of cells. In
particular, fluorescent probes have been developed to locally
measure the concentrations of important metabolites, such as
ions that are involved in signaling and energy transduction.
Despite their significance in many essential biological pro-
cesses, protons have not been studied as much as other ions
(e.g. Ca2+) by fluorescence microscopy;[8] a possible explan-
ation for this is the absence of entirely satisfactory fluorescent
indicators to measure pH in the relevant range (4.5–7.4).[9] In
fact, pH does not significantly alter the emission spectra of
most current fluorescent indicators, for instance, fluorescein
[*] S. Charier, Dr. O. Ruel, Dr. J.-B. Baudin, D. Alcor, Dr. J.-F. Allemand,
A. Meglio, Prof. Dr. L. Jullien
DØpartement de Chimie (C.N.R.S. U.M.R. 8640)
École Normale SupØrieure
24, rue Lhomond, 75231 Paris Cedex 05 (France)
Fax : (+33)1-4432-3325
E-mail: Ludovic.Jullien@ens.fr
Dr. J.-F. Allemand
DØpartement de Physique (C.N.R.S. U.M.R. 8550)
École Normale SupØrieure
24, rue Lhomond, 75231 Paris Cedex 05 (France)
Angew. Chem. Int. Ed. 2004, 43, 4785 –4788
DOI: 10.1002/anie.200460557
ꢀ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4785

Communications
and pyranin, other than to modify the intensity of their
fluorescence emission. To measure pH by fluorescence
spectroscopic techniques is a delicate process and requires
preliminary calibrations. Herein, we report a new fluorescent
indicator designed to measure pH in the range of 5–7 in
aqueous solution by means of a ratiometric method that relies
on fluorescence emission after one- and two-photon excita-
tion.
larger pKa value (ꢀ 8)[15] was sensitive to interference from
solutes other than protons.[16] Our design relied on the
introduction of substituents on the pyridine ring of 1 to
induce a shift in its pKa value without altering too strongly its
favorable photophysical features or its solubility in water. The
2-aminopyridyl derivative 2 (PYMPON) was hence pre-
pared—the pKa value of 2-aminopyridine is 6.9 at 208C
whereas that of pyridine is 5.3 at the same temperature.[17]
PYMPON 2 was obtained in three steps (Scheme 1): The
oxazole 5 was prepared in 88% yield by the condensation of
4-methoxyphenacylamine (3) with 2-chloroisonicotinic acid
To measure pH by a ratiometric fluorometric method,
both the acidic and basic states of the indicator should ideally
be fluorescent and exhibit shifted absorption and emission
spectra. In principle, this is the case when either the electron-
donor or the -acceptor moiety of a donor–acceptor fluoro-
phore exhibits acid or base properties. However, in practice,
numerous pathways compete with fluorescence emission for
the relaxation of the excited state in donor–acceptor chro-
mophores; for example, basic donor amino groups can act as
fluorescence quenchers.[6,7] An important constraint is thus to
have similar quantum yields of fluorescence for the
acidic and basic states of the pH indicator. Further-
more, the pKa values of the ground and excited states
are prone to be significantly different in many donor–
acceptor molecules owing to the redistribution of the
electron density about the molecule upon light
absorption. This feature may complicate the analy-
sis.[10–12]
A
2-(4-pyridyl)-5-aryloxazole backbone was
chosen to engineer the donor–acceptor fluorescent
pH probe. A series of oxazole derivatives was
originally investigated to develop laser dyes[13] owing
to the favorable photophysical features exhibited after
one-photon excitation: e ꢀ 3 ” 104 mꢁ1 cmꢁ1 at l ꢀ
350 nm and strong fluorescence emission (FF ꢀ 1) at
l ꢀ 500 nm. 2-(4-pyridyl)-5-(4-methoxyphenyl)oxa-
Scheme 1. Pathway for synthesis of the target compound PYMPON 2. a) POCl3,
D, 8 h; b) NH2NH2, H2O, D, 16 h; c) Raney Ni, EtOH, H2 (1 bar), room temper-
ature.
zole (PYMPO, 1) has already been reported to be an
attractive fluorescent probe to measure pH,[8,14] but its use is
restricted to the most acidic cellular compartments as it has a
low pKa value (pKa ꢀ 4). A related boronic acid probe with a
(4) and was subsequently converted into the hydrazine 6 in
41% yield. Reduction of the hydrazine moiety in the latter
compound yielded the aniline derivative 2 (PYMPON) in
51% yield. Figure 1 displays the absorption and emission
Figure 1. Photophysical properties of PYMPON 2 (in Britton–Robinson buffer)[20] at different pH values at 298 K; a) single-photon absorption
(molar absorption coefficient e in mꢁ1 cmꢁ1; solid line=pH 8, dotted line=pH 3) and two-photon absorptivity (TPA, =pH 9, =pH 2) spectra;
b) normalized steady-state fluorescence emission IF after one-photon excitation at l=335 nm (solid line=PYMPON 2, dotted line=2H+).
1 GM=10ꢁ50 cm4 s(photonmolecule)ꢁ1
*
*
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Angewandte
Chemie
spectra of 2, as well as its excitation spectra after two-photon
excitation at pH 3 and pH 8. PYMPON 2 exhibits a large one-
photon absorption band (e ꢀ 2 ” 104 mꢁ1 cmꢁ1) at lmax = 355 nm
(pH 3) and at lmax = 326 nm (pH 8); (Figure 1a). The bath-
ochromic shift observed upon decreasing the pH value is in
line with the increase in the electron-withdrawing power of
the pyridine ring upon protonation of the pyridyl nitrogen
atom. PYMPON is highly fluorescent at 298 K (Figure 1b):
At pH 3, the fluorescence emission maximum of the proto-
nated PYMPON species 2H+ is located at 530 nm with a
quantum yield FF = 0.7. At pH 8, the emission maximum of 2
lies at 465 nm with FF = 0.8.
Figure 1a also displays the two-photon excitation spectra
of PYMPON 2 recorded at pH 2 and 9 at 298 K. The power-
squared dependence of two-photon-excited fluorescence was
checked at several wavelengths in the investigated range and
revealed satisfactory behavior over the 0–60 mW range. The
two-photon absorption spectra compared well with the one-
photon absorption spectra after division of the wavelength by
a factor of 2: This suggests that the same excited states are
reached regardless of the excitation mode. Such an observa-
tion is in agreement with other reports that make use of a
comparable technique with unsymmetrical donor–acceptor
compounds.[18,19] In the present case with 2, the maximum
two-photon absorptivity at 710 nm (dmax(710)) is 60 ꢂ 10 GM
at pH 2 and 15 ꢂ 3 GM at pH 9 (1 GM = 10ꢁ50 cm4 s(photon-
molecule)ꢁ1). These values compare well with the corre-
sponding values for commonly employed pH probes (fluo-
rescein at pH 11: dmax(780) = 35 GM, FF = 0.9; pyranin back-
bone: dmax(750) = 4 GM, FF = 0.54[19]).
shifted by + 1.7 units relative to that of PYMPO
(pKa(1H+) ꢀ 4): this difference is similar to the corresponding
shift in the pyridine series (pKa(pyridine) = 5.3, pKa(2-amino-
pyridine) = 6.9[17]). Furthermore, one anticipates much lower
pKa values for acids that result from protonation of either the
nitrogen atom of the oxazole ring (pKa ꢀ 0) or the amino
group on the pyridine ring (pKa < 0).[17]
PYMPON Hþ Ð PYMPONþHþ
ð1Þ
Figure 2b shows that PYMPON 2 can serve as a
fluorescent probe to measure pH values in the range 3–8 by
a ratiometric method by tuning the excitation and emission
wavelengths [Eq. (3), see Experimental Section]. When one-
photon excitation is performed at the isobestic point
e,1
550=430
(339 nm), the ratio 1
of the fluorescence emissions at
550 nm and at 430 nm varies by one order of magnitude in the
e,2
550=430
pH 3.5–6.5 range. The same behavior is observed for 1
after two-photon excitation at 712 nm between pH 4.5–7.5.
In conclusion, the present study suggests that the
PYMPON platform 2 is appropriate to measure pH values
in the range 3–8 by a ratiometric method that relies on
fluorescence emission. This is especially attractive when two-
photon excitation is used as it would allow local addressing on
the femtoliter scale. Its similarity to the PYMPO backbone 1,
which was used to design an efficient probe,[8] suggests that
derivatives of PYMPON 2 could find useful applications as
intracellular pH meters.
The evolution of the fluorescence emission after one-
photon excitation of an aqueous solution of PYMPON 2
(100 nm) as a function of pH is shown in Figure 2. The acid–
base reaction depicted in Equation (1) yields pKa(2H+) =
5.7 ꢂ 0.1 at 298 K; the same value was extracted from the
pH-dependence of the one-photon absorption spectrum. The
pKa value confirms the earlier observation from the photo-
physical studies, that is, protonation of PYMPON 2 occurs at
the nitrogen atom of the pyridine ring. The pKa(2H+) value is
Experimental Section
1
3
2: H NMR (400 MHz, CDCl3, 258C, TMS): d = 8.19 (d, J = 5.4 Hz,
1H), 7.65 (AA’XX’, 3J = 8.8 Hz, 2H), 7.36 (s, 1H), 7.29 (dd, 3J =
5.4 Hz, 4J = 1.4 Hz, 1H), 7.16 (m, 1H), 6.98 (AA’XX’, 3J = 8.8 Hz,
2H), 3.87 ppm (s, 3H); 13C NMR (100.6 MHz, CDCl3, 258C, TMS):
d = 160.19, 158.88, 158.61, 152.27, 148.90, 136.07, 126.02, 122.34,
120.34, 114.48, 110.55, 104.65, 55.40 ppm; HRMS: calcd for
C15H14N3O2: 268.1086; found: 268.1084.
UV/Vis absorption and fluorescence spectra were recorded on a
Kontron Uvikon-940 spectrophotometer and a Photon Technology
International LPS 220 spectrofluorimeter,
respectively. The two-photon excitation
spectra were recorded with a homebuilt
setup[18] by using the reported excitation
spectrum of fluorescein for calibration.[19]
All experiments were performed at 293 K
in Britton–Robinson buffer solution
(0.1 molLꢁ1) prepared according to the
literature.[20]
Theoretical expressions of the ratios of
the absorbances and of the fluorescence
emissions at two wavelengths with pH:
Denoting AH for PYMPONH+ and A for
i
PYMPON, the ratio 1al ,i of the absorban-
1,l2
ces of PYMPON solutions at two different
wavelengths l1 and l2 after one (i = 1, ai =
Figure 2. a) Dependence of the emission spectrum of PYMPON 2 (100 nm in Britton-Robinson
buffer, 298 K) on pH: from acidic to basic conditions: pH 3.4, 4.5, 4.8, 5.1, 5.5, 6.0, 6.2, 6.5, 6.7,
e) or two (i = 2, ai = d) photon excitation
can be written:
7.0, 7.3, 9.1. b) Evolution of the ratio of the fluorescence emissions at 550 and 430 nm after one-
e,1
550=430
(1
, lexc =339 nm; dotted line) and two- (1e5,520=430, lexc =712 nm; solid line) photon excitations.
i
i
aAHðl1Þ10ꢁpH þ aAðl1Þ10ꢁpK
a
a
*
Experimental data: ; theoretical predictions: dotted and solid lines (calculated from Eq. (3) by
using the parameter values measured during the present study).
i
1al ,i
¼
ð2Þ
i
i
1,l2
aAHðl2Þ10ꢁpH þ aAðl2Þ10ꢁpK
Angew. Chem. Int. Ed. 2004, 43, 4785 –4788
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Communications
i
At a given excitation wavelength lexc, the ratio 1e ,i of the
l1,l2
fluorescence emissions of PYMPON solutions at two different
wavelengths l1 and l2 can be written:
i
a
i
IAH ðl1ÞꢀAH aAHðlexcÞ10ꢁpH þ IA ðl1ÞꢀA aAi ðlexcÞ10ꢁpK
*
*
1el ,i
¼
ð3Þ
*
*
1,l2
i
IAH ðl2ÞꢀAH aAHðlexcÞ10ꢁpH þ IA ðl2ÞꢀA aAi ðlexcÞ10ꢁpK
a
*
*
*
*
The terms f and I(l) designate the quantum yield of fluorescence
and the normalized emission spectrum of the considered species,
1
R
respectively ( I(l)dl = 1). The experimental curves were analyzed
0
with Specfit software to extract the pKa values.
Received: May 5, 2004
Keywords: acidity · basicity · fluorescent probes ·
.
nitrogen heterocycles · sensors
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4788
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