Rational design, synthesis, and spectroscopic and photophysical properties of a visible-light-excitable, ratiometric, fluorescent near-neutral pH indicator based on BODIPY

Article abstract of DOI:10.1002/chem.201002280

A visible-light-excitable, ratiometric, brightly fluorescent pH indicator for measurements in the pH range 5-7 has been designed and synthesized by conjugatively linking the BODIPY fluorophore at the 3-position to the pH-sensitive ligand imidazole through

Full text of DOI:10.1002/chem.201002280

DOI: 10.1002/chem.201002280
Rational Design, Synthesis, and Spectroscopic and Photophysical Properties
of a Visible-Light-Excitable, Ratiometric, Fluorescent Near-Neutral pH
Indicator Based on BODIPY
Noꢀl Boens,*[a] Wenwu Qin,[a, b] Mukulesh Baruah,[a] Wim M. De Borggraeve,[a]
Aleksander Filarowski,[a, c] Nick Smisdom,[d] Marcel Ameloot,[d] Luis Crovetto,[e]
Eva M. Talavera,[e] and Jose M. Alvarez-Pez*[e]
Abstract: A visible-light-excitable, ra-
tiometric, brightly fluorescent pH indi-
cator for measurements in the pH
range 5–7 has been designed and syn-
thesized by conjugatively linking the
BODIPY fluorophore at the 3-position
to the pH-sensitive ligand imidazole
through an ethenyl bridge. The probe
is available as cell membrane permea-
ble methyl ester 8-(4-carbomethoxy-
phenyl)-4,4-difluoro-3-[2-(1H-imidazol-
4-yl)ethenyl]-1,5,7-trimethyl-3a,4a-
diaza-4-bora-s-indacene (I) and corre-
sponding water-soluble sodium carbox-
ylate, sodium 8-(4-carboxylatophenyl)-
4,4-difluoro-3-[2-(1H-imidazol-4-yl)e-
thenyl]-1,5,7-trimethyl-3a,4a-diaza-4-
bora-s-indacene (II). The fluorescence
quantum yield Ff of ester I is very high
(0.8–1.0) in the organic solvents tested.
The fluorescence lifetime (ca. 4 ns) of I
in organic solvents with varying polari-
ty/polarizability (from cyclohexane to
acetonitrile) is independent of the sol-
vent with a fluorescence rate constant
kf of 2.4ꢀ108 sꢀ1. Probe I is readily
loaded in the cytosol of live cells,
where its high fluorescence intensity
remains nearly constant over an ex-
tended time period. Water-soluble indi-
cator II exhibits two acid–base equili-
bria in aqueous solution, characterized
by pKa values of 6.0 and 12.6. The Ff
value of II in aqueous solution is high:
0.6 for the cationic and anionic forms
of the imidazole ligand, and 0.8 for
neutral imidazole. On protonation–de-
protonation in the near-neutral pH
range, UV/Vis absorption and fluores-
cence spectral shifts along with isosbes-
tic and pseudo-isoemissive points are
observed. This dual-excitation and
dual-emission pH indicator emits in-
tense green-yellow fluorescence at
lower pH and intense orange fluores-
cence at higher pH. The influence of
ionic strength and buffer concentration
on the absorbance and steady-state
fluorescence of II has also been investi-
gated. The apparent pKa of the near-
neutral acid–base equilibrium deter-
mined by spectrophotometric and fluo-
rometric titration is nearly independent
of the added buffer and salt concentra-
tion. In aqueous solution in the ab-
sence of buffer and in the pH range
5.20–7.45, dual exponential fluores-
cence decays are obtained with decay
time t1 =4.3 ns for the cationic and t2 =
3.3 ns for the neutral form of II. The
excited-state proton exchange of II at
near-neutral pH becomes reversible on
ꢀ
addition of phosphate (H2PO4 /
HPO42ꢀ) buffer, and a pH-dependent
change of the fluorescence decay times
is induced. Global compartmental anal-
ysis of fluorescence decay traces col-
lected as a function of pH and phos-
phate buffer concentration was used to
recover values of the deactivation rate
constants of the excited cationic (k01 =
2.4ꢀ108 sꢀ1) and neutral (k02 =3.0ꢀ
108 sꢀ1) forms of II.
Keywords: dyes/pigments · fluores-
cent probes · pH indicators · photo-
physics · sensors
[a] Prof. N. Boens, Prof. W. Qin, Dr. M. Baruah,
Prof. W. M. De Borggraeve, Prof. A. Filarowski
Department of Chemistry
[e] Dr. L. Crovetto, Prof. E. M. Talavera, Prof. J. M. Alvarez-Pez
Department of Physical Chemistry
University of Granada, Cartuja Campus
Granada 18071 (Spain)
Katholieke Universiteit Leuven
Celestijnenlaan 200f - bus 02404, 3001 Leuven (Belgium)
E-mail: Noel.Boens@chem.kuleuven.be
E-mail: jalvarez@ugr.es
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201002280. It contains solvato-
chromism of absorption (n¯abs) and emission (n¯em) maxima of I; ratio-
metric fluorometric titrations of II for the estimation of Ka; pH de-
pendence of the excitation and emission spectra of II (pH 10.75–
12.54); pH dependence of fluorescence quantum yields of II; loading
of UBimi in live HEK 293 cells; photostability and retention of
UBimi in live HEK 293 cells; theory; experimental details; global
compartmental analysis results of time-resolved fluorescence of II;
[b] Prof. W. Qin
Key Laboratory of Nonferrous Metal Chemistry and Resources
Utilization of Gansu Province and State Key Laboratory
of Applied Organic Chemistry
College of Chemistry and Chemical Engineering
Lanzhou University, Lanzhou 730000 (P. R. China)
[c] Prof. A. Filarowski
Faculty of Chemistry, University of Wroclaw
F. Joliot-Curie 14, 50-383 Wrocław (Poland)
1
and H NMR spectrum of I.
[d] N. Smisdom, Prof. M. Ameloot
BIOMED, Universiteit Hasselt and transnational University Limburg
Agoralaan, Gebouw C, 3590 Diepenbeek (Belgium)
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FULL PAPER
Introduction
The design, synthesis, and spectroscopic/photophysical char-
acterization of novel fluorescent chemosensors continue to
be vibrant research topics.[1] Measurement of pH by fluores-
cence-based techniques is well established for both imaging
and sensing applications.[2,3] Fluorescent near-neutral pH in-
dicators that can quantify minor pH changes are especially
attractive targets in molecular design and synthesis.
Figure 1. Chemical structure of UBimi: methyl ester I and carboxylate
salt II.
Because of their excellent characteristics, 4,4-difluoro-4-
bora-3a,4a-diaza-s-indacene[4,5] (better known as BODIPY,[2]
difluoroboron dipyrromethene) derivatives are favored fluo-
rophores in the design of fluorescent probes. Indeed, the
valuable qualities of BODIPY[4,5] comprise relatively high
molar absorption coefficients and fluorescence quantum
yields, narrow emission bandwidths with high peak intensi-
ties, robustness towards light and chemicals, and excitation/
emission wavelengths in the visible spectral range (above
500 nm). Moreover, the spectroscopic properties of
BODIPY derivatives can be fine-tuned by synthetically in-
troducing suitable substituents at the right positions of the
difluoroboron dipyrromethene core.
sured by spectrophotometric and fluorometric titrations is
investigated. Finally, the excited-state kinetics of II in aque-
ous solution is investigated by global compartmental analy-
sis of the fluorescence decay surface, collected as a function
of emission wavelength, pH, and buffer concentration. The
major purpose of this work is to demonstrate that it is feasi-
ble, by judicious choice of fluorophore and ligand, to ration-
ally design a near-neutral fluorescent pH probe which has
many desirable properties. Note that intracellular pH meas-
urements are beyond the scope of this paper.
Difluoroboradiaza-s-indacenes with hydroxyaryl subunits
have been reported as pH indicators in aqueous–organic
mixed media.[6,7] These on/off pH indicators showed photo-
induced electron transfer (PET) from the twisted and conju-
gatively uncoupled phenolate to the BODIPY subunit, caus-
ing fluorescence quenching at high pH. 8-(4’-Hydroxyphen-
yl)-substituted BODIPY derivatives[6] sense the alkaline pH
range, while the tetrahydroxylated calix[4]arene derivative[7]
is sensitive in the near-neutral pH range. An on/off 8-(4’-di-
methylaminophenyl)-substituted BODIPY indicator has
been reported for the acidic pH range.[8,9] Quenching by
PET from the dimethylanilino group to the fluorophore ac-
counts for quenching of emission in the uncharged form.
Previously, we synthesized and spectroscopically character-
ized two water-soluble, on/off pH indicators with pKa
around 7.5 based on o-chlorophenol linked to the meso (8-)
position[10] or through a vinyl bridge to the 3-position[11] of
difluoroboron dipyrromethene. However, these indicators
showed substantial amplification of fluorescence intensity at
lower pH, without fluorescence spectral shift. A series of
pH probes based on BODIPY with meso-anilino substitu-
ents have been described recently for imaging acidic endo-
somes in cancer cells.[12] These derivatives are almost non-
fluorescent in basic media due to PET.
Here we describe the molecular design and synthesis of
UBimi (ultra-bright imidazole-based indicator), that is,
methyl ester I and its associated water-soluble sodium salt
II, substituted with a 2-(1H-imidazol-4-yl)ethenyl group at
the 3-position of the BODIPY core (Figure 1). We also in-
vestigate its solvent- and pH-dependent spectroscopic and
photophysical properties. Furthermore, we explore the
ground-state equilibrium between the protonated and neu-
tral imidazole forms of II through spectrophotometric and
fluorometric titrations. Next, the influence of the addition of
salt and buffer on the apparent acidity constant of II mea-
Results and Discussion
Design of the fluorescent pH indicator: Robustness of the
indicator against chemicals and light, a ground-state pKa
value close to the actual pH one intends to measure, excita-
tion and emission spectra in the visible wavelength range,
intense fluorescence (requiring large molar absorption coef-
ficients eACTHNUGTRNEG(UN lex) at the excitation wavelength lex and high fluo-
rescence quantum yields Ff, i.e., high dye brightness),[13] and
spectral shifts of the excitation and/or emission spectrum on
proton binding are all looked-for properties of the optimal
fluorescent pH indicator.
We chose imidazole/imidazolium with its favorable pKa of
7.0 to serve as pH-sensitive ligand for the near-neutral pH
range. Substitution of imidazole generally leads to different
pKa values. Due to the electron-withdrawing character of
BODIPY, it is expected that conjugatively linking the
BODIPY fluorescent reporter subunit via an ethenyl bridge
to imidazole will decrease the pKa of imidazole in UBimi by
approximately 1 unit compared to free imidazole. Imidazole
itself exhibits two acid–base equilibria: between the cationic
and neutral species with pKa =7 and between the neutral
and anionic forms with pKa =14.[14] We discuss this in detail
below for pH indicator II.
Because of its many excellent properties, we selected
BODIPY as key fluorophore for the novel pH indicator.
Owing to its beneficial properties, many probe requirements
are automatically fulfilled: (photo)chemical stability, bright
fluorescence due to high eACHTUNRGTEN(UNG lex) and Ff, and excitation with
visible light are all intrinsic to the BODIPY platform. The
fluorescent probe UBimi entails conjugation of the imida-
zole pH-sensing subunit through an ethenyl linker to the
BODIPY core at the 3-position. Based on the spectroscopic
properties of BODIPY derivatives with ethenylphenyl sub-
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N. Boens, J. M. Alvarez-Pez et al.
stituents at the 3- (and 5-) position(s),[15] it is expected that
conjugation of the imidazole ligand through an ethenyl
linker at the 3-position will introduce a pronounced batho-
chromic shift of both absorption (excitation) and emission
maxima compared to classic BODIPY fluorophores,[16] and
UBimi (II, Figure 1) was obtained by saponification with
NaOH.
Spectroscopic properties of I: Solvatochromism of UBimi
ester I was investigated in several solvents. Figure 2 shows
will lead to elevated photostability,[17] high Ff and e
ACTHNUTRGEN(UNG lex)
values, and may produce spectral shifts on protonation–de-
protonation. The pH-sensitive ligand is linked to the fluoro-
phore by a condensation reaction of commercially available
1H-imidazole-4-carbaldehyde with a methyl group at the 3-
position of BODIPY.[15] To increase the quantum yield Ff,
we chose
a 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-
diaza-s-indacene analogue, because there is evidence that
1,3,5,7-tetramethyl-substituted boradiazaindacene has high
Ff values.[5,10,18] To ensure cell membrane permeability, a
benzoate ester (in I), which can be converted to a water-
soluble carboxylate salt (in II), either by saponification or
by nonselective intracellular esterases, was introduced at the
meso position (Figure 1). The p systems of BODIPY and an
aromatic subunit at the meso position are highly twisted and
hence electronically essentially decoupled (i.e., no conjuga-
tion is possible between the BODIPY core and its aromatic
meso substituent).[8,19] Therefore, locating methyl benzoate
at the meso position will minimally affect the spectral (UV/
Vis absorption and emission) positions when it is converted
to an ionic carboxylate group. Likewise, fluorescent pH indi-
cators based on difluoroboron dipyrromethene in which a
proton signaling subunit connected to the meso position is
uncoupled from the fluorophore are never ratiometric.[6–10]
An essential design principle for a ratiometric, fluorescent
pH signaling system is to have conjugation between the pH-
(i.e., H+-) sensitive subunit and the fluorophore.
Synthesis of UBimi: 8-(4-Carbomethoxyphenyl)-4,4-di-
fluoro-3-[2-(1H-imidazol-4-yl)ethenyl]-1,5,7-trimethyl-3a,4a-
diaza-4-bora-s-indacene (I, Figure 1) was synthesized in
51% yield by condensation of 8-(4-carbomethoxyphenyl)-
4,4-difluoro-1,3,5,7-tetramethyl-3a,4a-diaza-4-bora-s-inda-
cene (III) with 1H-imidazole-4-carbaldehyde (IV) with
acetic acid/piperidine as catalyst (Scheme 1). Starting mate-
rial III is a known compound and was prepared from methyl
4-formylbenzoate and 2,4-dimethylpyrrole by following a lit-
erature procedure.[11] The sodium carboxylate form of
Figure 2. a) Absorption spectra of I in different solvents normalized to
1.0. b) Corresponding normalized fluorescence emission spectra (excita-
tion at lex =530 nm). c) Corresponding normalized excitation spectra (ob-
served at lem =660 nm).
Scheme 1. Synthesis of compound I. Conditions: i) toluene, piperidine,
AcOH, molecular sieves, reflux, 30 min, 51%.
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Fluorescent pH Indicator Based on BODIPY
FULL PAPER
the normalized UV/Vis absorption and fluorescence excita-
tion and emission spectra of I in various solvents. The ab-
sorption spectra are of similar shape to those of classic
BODIPY dyes,[5,15,16,18] with an intense absorption band with
maximum (corresponding to eACHTUNGTERN(NUG lex)=emax) in chloroform,
ethyl acetate, and methanol were 3.57ꢀ104, 4.36ꢀ104, and
3.91ꢀ104 Lmolꢀ1 cmꢀ1, respectively. To put these favorable
values into perspective, we compare them with representa-
tive literature values for some fluorescein and rhodamine-
based pH indicators useful at near-neutral pH. 2’,7’-Bis(2-
carboxyethyl)-5-(and -6-)carboxyfluorescein (BCECF) is
currently the most popular fluorescent indicator for estimat-
ing intracellular, near-neutral pH.[23,24] The phenolic form of
BCECF has emax =3.47ꢀ104 Lmolꢀ1 cmꢀ1 at 484 nm.[24] Since
no Ff data for BCECF in acidic solution are available, its
brightness cannot be determined. The corresponding pheno-
late form has redshifted absorption spectra with increased
molar absorption coefficients (emax =8.73ꢀ104 Lmolꢀ1 cmꢀ1
at 503 nm).[24] In combination with Ff =0.84,[25] this yields a
maximal brightness of 7.33ꢀ104 Lmolꢀ1 cmꢀ1. 2’,7’-Bis(2-car-
boxypropyl)-5-(and -6-)carboxyfluorescein (BCPCF) is a ho-
mologue of BCECF.[25] As expected, it has similar pKa
values, absorption and emission maximum wavelengths, and
Ff values. Seminaphthorhodamine derivative 5-(and 6-)car-
boxy-SNARF-1 (C.SNARF-1)[2,26] is probably the second
most widely used indicator for intracellular, near-neutral pH
a maximum labsACHTUNGTRENNUNG(max) located between 562 nm (in methanol)
and 575 nm (in toluene), assigned to the 0–0 band of the
!
S1 S0 transition, and a (more or less pronounced) shoulder
on the high-energy side, attributed to the 0–1 vibrational
band of the same transition. The labsACTHNUTRGNEUNG(max) values are hardly
affected by solvent polarity/polarizability, consistent with
the absorption spectroscopic behavior of common BODIPY
chromophores.[16,20] The solvent dependence of n¯abs [=1/labs
(max)] is illustrated in Figure S1 (Supporting Information).
In addition, a weaker, broad absorption band, attributed to
!
the S2 S0 transition, is found around 380 nm, the position
of which is practically independent of solvent. Compared to
classical BODIPY derivatives, labsACHTUNTRGNEUNG(max) is redshifted by ap-
proximately 50–60 nm.[5,15,16,18,20] UBimi in cyclohexane,
THF, and methanol is effectively optically transparent be-
tween 430 and 450 nm. The full width at half-height of the
maximum of the main absorption band (fwhmabs) is narrow
and nearly independent of solvent [(84ꢁ7)ꢀ10 cmꢀ1]. The
molar absorption coefficients emax at the absorption maxi-
measurements. The phenolic form of C.SNARF-1 has emax
=
mum labs
G
2.7ꢀ104 Lmolꢀ1 cmꢀ1 at 548 nm with Ff =0.03,[2,3,26] yielding
a brightness of only 810 Lmolꢀ1 cmꢀ1. Its corresponding phe-
nolate form has emax =4.8ꢀ104 Lmolꢀ1 cmꢀ1 at 576 nm with
Ff =0.09,[2,3,26] resulting in an increased, albeit low, bright-
ness of 4320 Lmolꢀ1 cmꢀ1. The phenolic form of 5-(and 6-)
carboxy-SNAFL-1 (C.SNAFL-1, a seminaphthofluorescein
derivative)[2,26] has emax =2.9ꢀ104 Lmolꢀ1 cmꢀ1 at 508 nm
compiled in Table 1 together with other relevant spectro-
scopic and photophysical data.
In all solvents studied, compound I also exhibits typical
BODIPY emission features (Figure 2), that is, a narrow,
slightly Stokes-shifted band of mirror-image shape.[16] The
maximum emission wavelength lem
589 nm range. The near-independence of the Stokes shift Dn¯
[=1/labs
with Ff =0.32,[2,3,26] resulting in
a
brightness of
(max)ꢀ1/lem
9280 Lmolꢀ1 cmꢀ1. The labs
ACHTUNGTRENNUNG
vent indicates that there is no appreciable difference be-
tween the permanent dipole moments of ground and excited
associated phenolate form of C.SNAFL-1 are 540 nm, 5.2ꢀ
104 Lmolꢀ1 cmꢀ1, and 0.08,[2,3,26] respectively, yielding a lower
brightness of 4160 Lmolꢀ1 cmꢀ1.
states.[21,22] The solvent dependence of n¯em [=1/lem
ACTHNUTRGNEU(GN max)] is
shown in Figure S2 (Supporting Information). The fluores-
cence quantum yields Ff for I are very high in all solvents
studied (0.78ꢂFf ꢂ1.00). An important photophysical prop-
erty of a fluorescent sensor is its brightness,[13] defined as the
product of the fluorescence quantum yield Ff and the molar
Fluorescence decay of I in organic solvents: To study the
time-resolved fluorescence of I, fluorescence decay histo-
grams in different solvents were collected as a function of
emission wavelength. In all solvents used, the fluorescence
decay profiles could be described by a single-exponential
decay function. The lifetimes t estimated by single-curve
analysis were independent of
absorption coefficient eACHTNUTRGNE(NUG lex) at the excitation wavelength lex.
The brightness values of I calculated for the absorption
the observation wavelength lem
Simultaneous (i.e., global)
curve fitting of the fluorescence
decay surface measured as a
function of lem with one linked
.
Table 1. Spectroscopic/photophysical data of I in different solvents at 208C.
[b]
[g]
Solvent[a]
labs(max) emax
[nm]
lem(max) fwhmabs Dn¯[c]
Ff[d] t[e]
kf[f]
knr
[Lmolꢀ1 cmꢀ1
]
[nm]
[cmꢀ1
]
[cmꢀ1
]
[ns] [108 sꢀ1
]
[108 sꢀ1
]
toluene
chloroform
cyclohexane
575
572
573
588
790
859
979
801
809
824
796
385
505
474
506
512
580
463
1.00 3.68 2.7
0.00
0.18
0.59
0.13
0.31
0.28
0.16
(3.8ꢁ0.3)ꢀ104 589
0.93 3.96 2.3
0.78 3.76 2.1
0.95 3.77 2.5
0.88 3.86 2.3
0.88 4.23 2.1
0.94 3.84 2.4
t
value confirmed that the
589
588
decays are indeed mono-expo-
nential and do not depend on
the emission wavelength. The
results of the time-resolved
fluorescence experiments are
also listed in Table 1. The t
values of I are practically sol-
vent independent (t=3.9ꢁ
tetrahydrofuran 571
ethyl acetate
acetonitrile
methanol
568
563
562
(5.0ꢁ0.6)ꢀ104 585
582
(4.2ꢁ0.5)ꢀ104 577
[a] The solvents are ordered according to decreasing polarizability (as measured by the refractive index n).
[b] Molar absorption coefficients at labs(max). [c] Stokes shift Dn¯ =1/labs(max)ꢀ1/lem(max). [d] The standard
uncertainties of the fluorescence quantum yields Ff were determined to be in the range 1–10%. [e] The stan-
dard errors on all lifetimes t are ꢂ10 ps. [f] Fluorescence deactivation rate constant. [g] Nonradiative deactiva-
tion rate constant.
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N. Boens, J. M. Alvarez-Pez et al.
0.2 ns). The values of the fluorescence deactivation rate con-
stant kf, calculated according to Equation S15 (Supporting
Information) from Ff and the globally estimated t values,
are shown to be independent of the solvent used, with a
mean value of kf =(2.4ꢁ0.2)ꢀ108 sꢀ1. The rate constants of
radiationless deactivation knr, calculated according to Equa-
tion S16 (Supporting Information), are also compiled in
Table 1.
pH dependence of II: Imidazole in aqueous solution has
two pH-dependent acid–base equilibria, characterized by
pKa values of 7 (protonated–neutral imidazole) and 14 (neu-
tral–anionic imidazole).[14] Fluorescent probe II with an imi-
dazolyl group will show comparable behavior (Figure 3).
Figure 3. pH-dependent acid–base equilibria of the imidazole group of II.
Visible absorption spectroscopy of II in aqueous (buffer) so-
lution: Spectrophotometric and fluorometric titrations (vide
infra) show that UBimi II in aqueous solution has two pH
dependent acid-base equilibria, characterized by pKa values
around 6 (protonated–neutral) and 12.6 (neutral–anionic).
At near-neutral pH, only the neutral and cationic forms of
II are relevant (Figure 3). The visible absorption spectra of
aqueous solutions of II in the pH range between 5.44 and
8.12 in acetate buffer with different KCl concentrations and
in phosphate buffer with different KCl concentrations were
recorded. The experimental absorption spectra of II at dif-
ferent pH show pH-induced transitions in the pH regions
dictated by the ground-state pKa value. Figure 4a shows an
example of the visible absorption spectra recorded for II at
different pH values in 50 mm acetate buffer in the absence
of KCl. In slightly basic solution, the spectrum is composed
of a band characterized by a sharp maximum at 560 nm and
a small shoulder around 520 nm. When the pH decreases,
the peak at 560 nm is blueshifted to 547 nm, its absorbance
decreases, and the shoulder is blueshifted, too. In the pH
range 5.44–8.12, two isosbestic points can be clearly distin-
guished, at 530 and 545 nm. The experimental visible ab-
sorption spectra of II at pH values ranging between 5.44
and 8.12 show that one pH-induced transition at near-neu-
tral pH is involved. The isosbestic points are consistently
positioned at 530 and 545 nm, independent of the acetate or
phosphate concentration. Therefore, it is concluded that the
acetate or phosphate buffer does not significantly perturb
the absorption spectrum of aqueous solutions of II, because
II does not form ground-state complexes with these buffers.
Assuming the acid–base equilibria between the prototrop-
ic forms depicted in Figure 3, the apparent acidity constant
pKaapp of the cation–neutral equilibrium can be determined
Figure 4. a) Visible absorption spectra of II in 50 mm acetate buffer and
in the absence of KCl, at pH values between 5.44 and 8.12. b) Global
nonlinear least-squares curve fitting of Equation S17 (Supporting Infor-
mation) to the absorbance A560 at 560 nm versus pH. c) Recovered eC
&
*
(cationic 1, ) and eN (neutral 2, ) values of II in 50 mm acetate buffer
in the presence of 75 mm KCl. The values are normalized to the maxi-
mum value of eN.
by using the experimental absorbance changes. A typical
data surface of absorbance A versus pH versus labs at each
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Fluorescent pH Indicator Based on BODIPY
FULL PAPER
buffer concentration and ionic strength was composed of 12
pH values and 100 labs values between 450 and 650 nm.
Beerꢂs law for two prototropic species turned out to be the
best model to fit Equation S17 (Supporting Information) to
the experimental absorption data. A plot of the absorbance
A560 at 560 nm versus pH is shown in Figure 4b. In the data-
surface fitting process, the estimated parameters were inde-
pendent of the initial guesses assigned to these parameters.
Figure 4c shows the e(l) values of the cationic (eC =e1) and
neutral (eN =e2) forms of II, normalized by the maximum
value of eN, recovered from the fitting of the experimental
absorption data of II in 50 mm acetate buffer in the presence
of 75 mm KCl. These molar absorption coefficient ratios
were practically independent of the ionic strength. Since ab-
solute values of eC and eN cannot be obtained (see Prepara-
tion of II in Experimental Section), the brightness of the
cationic and neutral forms of II cannot be determined. The
blueshift of the absorption spectrum of the cationic versus
the neutral form of II is clearly visible.
pH dependence of the excitation and emission spectra of II
in the near-neutral pH region: Figure 5 displays the fluores-
cence excitation and emission spectra of UBimi II as a func-
tion of pH in the pH range 4.5–7.0 in aqueous buffer solu-
tion
[50 mm
3-(N-morpholino)propanesulfonic
acid
(MOPS)] at room temperature. The excitation spectra (Fig-
ure 5a) show a decrease of the excitation band at 556 nm
(pH 7.0) and a blueshift to 547 nm (pH 4.5) with increasing
H+ concentration (lower pH). Thus, the excitation band of
the cationic form of II is blueshifted by about 10 nm in com-
parison with that of the neutral form. A pseudo-isoemissive
point at about 430 nm can be observed. The acidity constant
Ka of the imidazolium–imidazole equilibrium of II
(Figure 3) and the stoichiometry of proton binding by neu-
tral imidazole were determined from direct fluorometric ti-
tration as a function of pH by fitting Equation S19 (Support-
ing Information) to the fluorescence excitation data F. The
results obtained at lex =556 nm (corresponding to the excita-
tion wavelength of the maximum intensity of the neutral
form) and lem =590 nm indicated a 1:1 stoichiometry and
yielded a value of 5.87ꢁ0.03 for pKa (inset of Figure 5a).
Because there is a shift of the excitation spectra, ratiometric
excitation measurements can be performed. A pKa value of
5.83ꢁ0.04 was estimated by fitting Equation S20 (Support-
ing Information) to the fluorescence excitation ratios R=F-
Figure 5. a) Fluorescence excitation spectra (observation wavelength
lem =590 nm) and b) emission spectra (excitation wavelength lex
=
ACHTUNGTRENNUNG
(lem,l1ex)/F(lem,le2x) at l1ex/l2ex =547/556 nm and lem =590 nm.
520 nm) of II in aqueous buffered solution (50 mm MOPS) as a function
of pH. The full lines in the insets of a) and b) show the best fits (with n=
1) to the direct excitation (a, Equation S19 of the Supporting Informa-
tion) and ratiometric emission (b, Equation S20 of the Supporting Infor-
mation) titration data of II as a function of [H+].
A similar data analysis of the ratios R, taking the wave-
length of the pseudo-isoemissive point as l2ex =430 nm with
l1ex =556 nm and lem =590 nm, yielded pKa =5.72ꢁ0.06.
Choosing the wavelength of the pseudo-isoemissive point as
l2ex leads to a value of 1 for z=Fmin(lem,le2x)/Fmax(lem,l2ex) in
Equation S20 (Supporting Information) for the excitation
ratiometric method. Nonlinear fits of Equation S20 (Sup-
porting Information) to the ratiometric excitation fluoro-
metric data of II in aqueous buffered solution in the pres-
ence of KCl are shown in Figure S3 (Supporting Informa-
tion).
The fluorescence emission spectra of II as a function of
pH (Figure 5b) show a shift of the maximum from 570
(pH 7.0) to 557 nm (pH 4.5) with a pseudo-isoemissive point
at 567 nm. The fluorescence quantum yield of the neutral
(i.e., imidazole) form (Ff =0.8) is higher than that of the
cationic (i.e., imidazolium) form (Ff =0.6); pH indicator II
Chem. Eur. J. 2011, 17, 10924 – 10934
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
10929

N. Boens, J. M. Alvarez-Pez et al.
Table 2. Apparent pKa values of UBimi II at 208C determined by ab-
sorbance (Equation S17, Supporting Information), and direct (Equa-
tion S19, Supporting Information) and ratiometric (Equation S20, Sup-
porting Information) fluorometric titrations in the absence/presence of
added buffer and salt (KCl). For the fluorometric titrations, between 5
and 12 pKa measurements were included in the calculation of average
and standard uncertainty for each experimental condition. The last two
entries refer to the neutral–anion equilibrium, and all the other entries to
the neutral–cation equilibrium.
emits intense green-yellow fluorescence at lower pH and in-
tense orange fluorescence at higher pH (Figure 6).
[b]
[c]
Experimental conditions
Buffer ([m]) KCl [m]
m[a]
pKa
pKa
0
0
0
0
0
0
0.1
0
0
5.87ꢁ0.07
5.9ꢁ0.1
6.03ꢁ0.06
5.91ꢁ0.09
5.88ꢁ0.09
MOPS (0.01)
MOPS (0.05)
acetate (0.05)
phosphate (0.05)
0
acetate (0.125)
phosphate (0.075)
phosphate (0.05)
acetate (0.1)
phosphate (0.175)
phosphate (0.175)
acetate (0.1)
acetate (0.1)
0
0.01
0.05
0.05
0.1
Figure 6. Photograph of the fluorescence emitted by the neutral (orange)
and cationic (green-yellow) forms of UBimi II due to excitation at
366 nm. The red arrow indicates where protonation takes place.
6.2ꢁ0.2
6.0ꢁ0.1
0.1
0.125
0.15
0.2
5.92ꢁ0.05
5.89ꢁ0.05
0
Direct and ratiometric fluorometric titrations were used
to estimate values for the stoichiometry of proton binding
and Ka. For example, direct emission fluorometric titration
according to Equation S19 (Supporting Information) at lex =
520 nm and lem =557 nm gave a pKa of 6.00ꢁ0.05. Con-
versely, a value of 5.93ꢁ0.02 was estimated from ratiometric
measurements to be l1em/le2m =557/570 nm, in excellent agree-
ment with previous values. Selecting the wavelength of the
pseudo-isoemissive point as l2em gives a value of 1 for z=
Fmin(lem,l2ex)/Fmax(lem,le2x) in Equation S20 (Supporting Infor-
mation) for the emission ratiometric method. A mean pKa
value of 5.9ꢁ0.1 was obtained from all direct and ratiomet-
ric fluorometric titrations by using excitation or emission
data of II in 50 mm MOPS buffered aqueous solution
(Table 2). This value matches that obtained by means of ab-
sorption measurements in aqueous 50 mm acetate buffer so-
lution (Table 2). These fluorometric and spectrophotometric
titration data show that substitution of imidazolium (with
pKa ꢃ7.0)[14] with a difluoroboron dipyrromethene subunit
lowers its pKa to about 6.0, that is, BODIPY is indeed elec-
tron-withdrawing. Similar effects on the pKa of phenol- and
naphthol-substituted BODIPY derivatives have been report-
ed.[5,10,11]
0.1
0.2
0
0.1
0.6
0.9
0
6.01ꢁ0.05
0.3
5.96ꢁ0.06
5.96ꢁ0.03
5.99ꢁ0.04
5.96ꢁ0.08
5.98ꢁ0.07
0.35
0.45
0.7
1.0
0
12.61ꢁ0.03
12.63ꢁ0.06
0
0.1
0.1
[a] Ionic strengths m were calculated for solutions at 208C and pH 6.8 for
phosphate buffer, pH 7.2 for MOPS buffer, and 4.76 for acetate buffer
(these pH values correspond to the pKa values of the respective buffers,
denoted by pKBa , at 208C). [b] Determined from spectrophotometric
measurements. [c] Determined from fluorometric measurements.
shows an example of nonlinear fit of Equation S20 (Support-
ing Information) to the ratiometric fluorescence emission
data of II at alkaline pH (10.75–12.54). Addition of 100 mm
KCl gave an almost identical average pKa value of 12.63ꢁ
0.06. Our fluorometric titration data indicate that when imi-
dazole (with pKa ꢃ14 in the alkaline pH range)[14] is substi-
tuted with BODIPY at the 3-position (as in II), its pKa
value drops by more than one unit (to 12.6), once more con-
firming the electron-withdrawing power of BODIPY. An
overall view of the pH dependence of Ff of II is shown in
Figure S6 (Supporting Information).
pH dependence of II in the alkaline pH region: Figure S4
(Supporting Information) displays the excitation and emis-
sion spectra of II as a function of pH (10.75–12.54) in pure
water (i.e., without added buffer or salt). The fluorescence
excitation spectra of II show a decrease of the band with a
maximum at 556 nm with increasing pH and a pseudo-isoe-
missive point at 430 nm. The emission spectra of II exhibit
two bands with maxima at about 545 and about 570 nm.
With increasing [H+] (lower pH), the 545 nm band remains
almost constant, while the 570 nm emission band increases
in intensity. Values for the acidity constant Ka of imidazole
(neutral form) were determined from direct fluorescence ex-
citation and emission data F (Equation S19, Supporting In-
formation) and R values from ratiometric emission data
(Equation S20, Supporting Information) at various excita-
tion/emission wavelengths, and yielded a median pKa value
of 12.61ꢁ0.03 (Table 2). Figure S5 (Supporting Information)
Influence of ionic strength on Kaapp of II: Since the ionic
strength affects the ratio of the concentrations of the cation-
ic and neutral forms, and hence the optical signal from
UBimi, a comprehensive study of the dependence of Kaapp on
the ionic strength m is of interest. To study the influence of
added buffer and salt on Kaapp of II, we recorded pH-depen-
dent UV/Vis absorption and fluorescence excitation and
emission spectra of II in aqueous solutions with different
ꢀ
concentrations of buffer (MOPS, acetate, and H2PO4 /
HPO42ꢀ) and KCl. The steady-state fluorescence spectra
were analyzed according to Equations S19 and S20 (Sup-
porting Information) to estimate Ka. For the global analysis
of the absorbance, Equation S17 (Supporting Information)
was used. The recovered pKaapp values of II at different ionic
strengths along with the associated standard uncertainties
are listed in Table 2. Increasing ionic strength m only slightly
10930
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Chem. Eur. J. 2011, 17, 10924 – 10934

Fluorescent pH Indicator Based on BODIPY
FULL PAPER
increases the apparent ground-state acidity constants pKaapp
up to m=0.15; thereafter, pKaapp is insensitive to ionic
strength. Thus, a minor effect of the ionic strength m on the
apparent acidity constant of II was observed in the m range
studied. Moreover, the chemical nature of the buffer is irrel-
evant. This effect of ionic strength on the apparent acidity
constants is in accordance with the Debye–Hꢃckel theory,
and is typical of compounds in which the electrolytic charge
decreases by the reaction of deprotonation.[27] The pKa
values obtained from fluorometric measurements agree ex-
cellently with those from spectrophotometric titrations. The
near-independence of pKaapp of II from ionic strength is a
great advantage in fluorescent pH indicators, in which a neg-
ligible sensitivity to ionic strength is desired. This negligible
sensitivity of II to ionic strength is in contrast to some com-
mercially available pH probes, such as BCECF.[24]
at pH 5.20, 6.82, and 7.45 at lem =560, 570, 580, and 590 nm
provided us with reliable decay time estimates: t1 =4.28ꢁ
0.04 ns and t2 =3.3ꢁ0.2 ns. Regardless of the initial {ti, ai}
guesses, global curve fitting of these eleven decay curves re-
sulted in the same parameter estimates with the same high
precision. The longer lifetimes of II are quite close to the
values of its corresponding ester form I (ca. 4 ns) in various
organic solvents (from cyclohexane to acetonitrile). To
change the decay times {t1, t2} and to obtain unique esti-
mates for all rate constants defining the photophysical
model in Scheme S1 of the Supporting Information (in other
words, to have a uniquely identifiable photophysical model),
ꢀ
phosphate buffer (H2PO4 /HPO42ꢀ) was added to the aque-
ous solution of II.[28] The fluorescence decay times {t1, t2} of
II at constant pH (6.82) as a function of phosphate buffer
concentration CB and emission wavelength lem were estimat-
ed by classical global analysis and are compiled in Table 3.
Table 4 lists the globally estimated {t1, t2} of II at constant
Time-resolved fluorescence of II in aqueous solution in the
absence/presence of added buffer: The fluorescence decay
traces of I in organic solvents were shown to be single-expo-
nential (see above). To test the possibility of applying II in
fluorescence lifetime imaging (FLIM) in aqueous environ-
ment, fluorescence decays were collected first for II in pure
water followed by decay experiments on addition of buffer.
To test the quality of the experimental time-resolved fluo-
rescence data of II, each individual fluorescence decay trace
measured as a function of emission wavelength lem, pH, and
analytical buffer concentration CB was first analyzed sepa-
rately by a bi-exponential function in terms of decay times
{t1, t2} and associated pre-exponential factors {a1, a2}. Such
single-curve analysis not only discloses the number of
needed exponential terms but also tests the quality of the
experimental decay data and allows weeding out of inferior
experimental decay data. Traces which gave unacceptable
fits as bi-exponentials were eliminated from further analysis.
Secondly, classic global bi-exponential analyses in terms
of ti and ai were performed by incorporating in single decay
surface curves collected at the same pH and CB, but at dif-
ferent lem. The decay times ti were linked (held in common)
for decay traces measured at different lem. A lot of the bi-
exponential fluorescence decays of II had an almost negligi-
ble contribution corresponding to the shorter decay time t2.
Consequently, in these cases, especially in single-curve anal-
ysis, unreliable estimates may be obtained for t2. It can be
expected that the standard global analysis will estimate the
{t1, t2} and {a1, a2} values with higher accuracy and precision
than single-curve analysis. According to the photophysical
model presented in Scheme S1 (Supporting Information),
the decay times {t1, t2} in the absence of added buffer
should be independent of lem and pH (within the near-neu-
tral range). Under these circumstances, we have t1 =(k01 +
k21)ꢀ1 and t2 =k0ꢀ21, that is, the photophysical model in the ab-
sence of added buffer is not identifiable.[28] (For the defini-
tion of the rate constants kij, see Scheme S1, Supporting In-
formation). Global bi-exponential analysis of the fluores-
cence decay surface (c2g =1.08) including eleven curves in
the absence of buffer (i.e., concentration of buffer CB =0m)
Table 3. Globally estimated biexponential decay times of II in aqueous
solution at constant pH (6.82) as a function of total phosphate buffer
concentration CB and emission wavelength lem
.
CB [m]
t1 [ns]
t2 [ns]
c2g[a]
0
4.28ꢁ0.04
4.19ꢁ0.04
4.06ꢁ0.02
4.05ꢁ0.02
4.18ꢁ0.02
3.3ꢁ0.2
1.05
1.04
1.05
1.06
1.10
0.05
0.10
0.15
0.25
3.3ꢁ0.2
2.4ꢁ0.3
2.37ꢁ0.09
2.23ꢁ0.06
[a] Equation S21, Supporting Information.
Table 4. Globally estimated bi-exponential decay times of II in aqueous
solution at constant phosphate buffer concentration CB (0.1m) as a func-
tion pH and lem
.
pH
t1 [ns]
t2 [ns]
c2g[a]
7.25
7.00
6.82
6.45
6.15
5.80
5.45
4.03ꢁ0.02
4.08ꢁ0.02
4.06ꢁ0.02
4.12ꢁ0.03
4.15ꢁ0.03
4.27ꢁ0.03
4.28ꢁ0.05
2.1ꢁ0.1
2.3ꢁ0.1
2.5ꢁ0.3
2.8ꢁ0.1
2.9ꢁ0.2
2.9ꢁ0.2
3.1ꢁ0.3
1.07
1.09
1.05
1.07
1.05
1.10
1.05
[a] Equation S21, Supporting Information.
CB (0.1m) as a function of pH and lem. In each global bi-ex-
ponential analysis reported in Tables 3and 4, fluorescence
decay histograms collected at several lem values were com-
bined and analyzed with common (linked) {t1, t2}. Global
compartmental analysis of II in aqueous solution as a func-
tion of pH, lem, and pH buffer concentration CB was finally
used to recover values of all rate constants (Table S1, Sup-
porting Information). The most relevant decay parameters
are the deactivation rate constants of the excited cationic
(k01 =2.4ꢀ108 sꢀ1) and neutral (k02 =3.0ꢀ108 sꢀ1) forms of II.
Fluorescence imaging microscopy of UBimi in HEK 293
cells: Water solubility and membrane permeability are cru-
cial properties for a fluorescent pH indicator that functions
Chem. Eur. J. 2011, 17, 10924 – 10934
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
10931

N. Boens, J. M. Alvarez-Pez et al.
inside live cells. To test whether UBimi fulfills these addi-
tional requirements, measurements on live cells were per-
formed. We chose HEK 293 cells because of their large
nuclei, which allow one to monitor loading and photo-
bleaching of the probe both in the nuclei and the cytosol.
The strategy of delivery of UBimi into cells uses diffusion of
the cell membrane permeable ester I into cells, where it is
hydrolyzed by nonselective intracellular esterases to afford
the charged, fluorescent dye II.
Based on the fluorescence intensity on excitation at
543 nm of the HEK 293 cells during dye loading (200 nm), it
can be concluded that UBimi efficiently accumulates inside
the cytosol, but is excluded from the cell nucleus (Figure 7).
escein) are not retained well by living cells and rapidly leak
out.[23]
Conclusion
Novel BODIPY-derived pH indicator UBimi, available as
methyl ester I and sodium salt II, for the near-neutral pH
range with bright fluorescence in the yellow to orange spec-
tral region was synthesized by connecting imidazole to the
3-position of the BODIPY platform through a vinyl bridge.
Steady-state and time-resolved fluorescence measurements
were employed to study the spectroscopic and photophysical
properties of I as a function of solvent. The fluorescence
lifetime (3.9ꢁ0.2 ns) and fluorescence rate constant (kf =
(2.4ꢁ0.2)ꢀ108 sꢀ1) of I are independent of the solvent. In
aqueous solution, the water-soluble dye II undergoes a re-
versible protonation–deprotonation reaction in the near-
neutral pH range that is responsible for the observed spec-
tral shifts of the excitation and emission spectra. The appar-
ent pKaapp of II in aqueous (buffer) solution was obtained by
means of absorbance and fluorometric titrations and is prac-
tically insensitive to low ionic strength. In aqueous solution
in the absence of buffer in the pH range 5.20–7.45, dual-ex-
ponential fluorescence decays were obtained with t1 =4.3 ns
for the cationic and t2 =3.3 ns for the neutral form of II.
Global compartmental analysis of the fluorescence decay
surface of II in aqueous solution as a function of lem, pH,
and the absence/presence of phosphate buffer enabled us to
estimate all the excited-state rate constants of II. The very
high Ff values of the neutral (0.8) and cationic (0.6) forms
of II in aqueous solution, the possibility of using longer exci-
tation/emission wavelengths, the pKa value of 6.0, and the
spectral shifts in excitation as well as emission spectra (i.e.,
dual excitation and dual emission) make this new BODIPY
chemosensor an excellent ratiometric fluorescent probe for
pH measurements in the pH range 5–7. Dual-emission ratio-
metric probes allow for fast measurements, because two
emission channels can be recorded simultaneously. Methyl
ester I is readily taken up by biological cells in the cytosol,
where it is highly fluorescent and adequately photostable.
Recalling the design criteria for the optimal fluorescent pH
indicator, one can conclude that building a fluorescent indi-
cator with numerous required properties is feasible, al-
though improvements should be implemented for the con-
struction of a fluorescent indicator useful for intracellular
pH sensing and imaging. For cytosolic pH measurements, a
better match between the pKa of the indicator and physio-
logical pH (6.8–7.4) is desirable. To increase the pKa value
of the current probe (6.0) one should replace the unsubsti-
tuted imidazole group by one with electron-releasing sub-
stituents. Although UBimi is retained well inside cells, ideal-
ly one may want to increase the number of ester functionali-
ties so that more negatively charged carboxylate groups will
be present after their transformation by endogenous esteras-
es. Development of pH chemosensors along these lines is
currently in progress.
Figure 7. HEK 293 cells stained with UBimi. HEK 293 cells were loaded
with 200 nm of Ubimi I for 30 min at 218C. After rinsing three times with
the physiological solution to remove the free probe, the pseudocolor
image was collected with a confocal microscope (543 nm excitation,
25.6 ms pixel dwell time, 512ꢀ512 pixels). The scale of relative intensities
is given on the right. UBimi clearly accumulates in the cytosol and not in
the cell nucleus, apart from some weak spots possibly indicating nucleoli.
Only small spots in these nuclei, probably nucleoli, display
some fluorescence. Bright spots in the cytosol can be noticed
and might indicate accumulation in organelles or intracellu-
lar vesicles. The efficient accumulation of UBimi also sug-
gests good membrane penetration, which makes the use of
pluronic acid obsolete. Images with sufficiently high signal-
to-noise ratio can be obtained at other excitation wave-
lengths (458, 488 nm, 514 nm). Figure S7 (Supporting Infor-
mation) shows the loading of UBimi in live HEK 293 cells.
The photostability and retention of UBimi in HEK 293
cells were monitored over about 20 min period under a con-
focal microscope. No significant change in fluorescence
signal of UBimi could be observed (Figure S8, Supporting
Information). This distinguishes UBimi from most fluores-
cein-based pH indicators such as BCECF, which bleach rela-
tively quickly.[29] Other fluorescein derivatives (e.g., fluores-
cein diacetate, 6-carboxyfluorescein, and 5,6-dicarboxyfluor-
10932
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Chem. Eur. J. 2011, 17, 10924 – 10934

Fluorescent pH Indicator Based on BODIPY
FULL PAPER
Talented Persons to Lanzhou University. W.Q. is supported by the Pro-
gram for New Century Excellent Talents in University (NCET-09-0444)
and the Fundamental Research Funds for the Central Universities (lzujb-
ky-2011-22). This study was supported in part by the Key Program of Na-
tional Natural Science Foundation of China (20931003). N.S. is supported
by a grant from the Instituut voor de aanmoediging van innovatie door
Wetenschap en Technologie in Vlaanderen (IWT). M.A. acknowledges
support from tUL-impuls. This work was supported in part by grant P07-
Experimental Section
General aspects: Details of instrumentation, materials, preparation of sol-
utions, steady-state UV/Vis absorption and fluorescence spectroscopy,
time-resolved fluorescence spectroscopy, data analysis of time-resolved
fluorescence, cell culture, and fluorescence imaging microscopy are given
in the Supporting Information.
Fluorescence decay kinetics and identifiability of a pH probe in the ab-
sence/presence of added buffer has been described previously.[28] Equa-
tions relevant for this study are presented in the Supporting Information.
FQM-3091 from the Consejerꢄa de Innovaciꢅn, Ciencia
(Junta de Andalucꢄa).
y Empresa
Details of the determination of kf and knr from t and Ff, determination
of Ka and the molar absorption coefficients ratio eC/eN from spectropho-
tometric titration, influence of ionic strength on pKaapp, and determination
of Ka from direct and ratiometric fluorometric titration are given in the
Supporting Information.
[1] a) Chemosensors of Ion and Molecule Recognition (Eds.: J.-P. Des-
vergne, A. W. Czarnik), Kluwer, Dordrecht, The Netherlands, 1997;
b) B. Valeur, Molecular Fluorescence. Principles and Applications,
Wiley-VCH, Weinheim, Germany, 2002.
Synthesis of 8-(4-carbomethoxyphenyl)-4,4-difluoro-3-[2-(1H-imidazol-4-
yl)ethenyl]-1,5,7-trimethyl-3a,4a-diaza-4-bora-s-indacene (I) (Scheme 1):
1H-Imidazole-4-carbaldehyde (IV, 12 mg, 0.13 mmol) was added to a so-
lution of III[11] (38 mg, 0.1 mmol) in toluene (5 mL). To this mixture, one
drop of piperidine (0.10 mL), one drop of acetic acid (0.10 mL), and a
small amount of a molecular sieve were added. The reaction mixture was
then heated to reflux for 30 min. After cooling, the crude reaction mix-
ture was purified by silica-gel column chromatography, eluting first with
dichloromethane until the starting compound was collected and then
changing to 2:1 (v/v) CH2Cl2/ethyl acetate to afford 21 mg (51%) of I as
a violet powder. M.p. the crystal first changes color and does not melt up
to 3108C; 1H NMR: (300 MHz, CDCl3) d=8.21 (d, 2H, J=8.4 Hz), 7.74
(s, 1H), 7.45 (d, 1H, J=15.5 Hz), 7.43 (d, 2H, J=8.2 Hz), 7.32 (s, 1H),
7.28 (d, 1H, J=16.2 Hz), 6.57 (s, 1H), 6.02 (s, 1H), 3.99 (s, 3H,
COOCH3), 2.59 (s, 3H), 1.41 (s, 3H), 1.38 ppm (s, 3H); 13C (75 MHz,
CDCl3): d=14.5, 14.7, 29.7, 52.3, 116.9, 117.8, 121.5, 128.7, 130.3, 130.9,
136.8, 139.9, 142.4, 153.1, 155.7, 161.9, 166.5 ppm; LRMS (EI, 70 eV): m/
z: 460 [M]+ (100), 440 (90); HRMS (EI, positive): m/z calcd for
C25H23BF2N4O2: 460.1882 [M]+, found 460.1876.
Preparation of II: To investigate the H+-binding properties of the new
indicator in aqueous solution, ester I was treated with excess base to
yield the corresponding water-soluble sodium salt II.[30] A methanolic so-
lution of I was mixed with a highly concentrated solution of 3.5 equiv of
spectroscopic-grade NaOH in Milli-Q water. The reaction mixture was
heated to reflux for 20h. The mixture was allowed to cool to RT, more
Milli-Q water was added, and the solution was washed with spectroscop-
ic-grade chloroform to extract any residual starting material and possible
free-base dipyrromethene side products.[31] The separated aqueous layer
was evaporated to dryness in a lyophilization apparatus. The lyophilized
product was used to prepare solutions of the indicator for further fluores-
cence measurements. The multistep procedure of converting ester I to
carboxylate salt II (reaction with excess base followed by extraction and
finally lyophilization) is not quantitative. Hence, the ultimate amount of
II in the lyophilized residue is unknown, therefore, it is impossible to
obtain absolute values of the absorption coefficients e(lex) of II, and the
brightness of the cationic and neutral forms of II cannot be determined.
Comparison of the absorption and fluorescence emission spectra of ester
I in methanol and sodium salt II in water indicates that the fluorophore
structure remains intact.
[2] R. P. Haugland, The Handbook. A Guide to Fluorescent Probes and
Labeling Technologies, 10th ed., Molecular Probes, Eugene, Oregon,
2005, pp. 935–955.
[3] J. Han, K. Burgess, Chem. Rev. 2010, 110, 2709–2728.
[4] A. Treibs, F.-H. Kreuzer, Justus Liebigs Ann. Chem. 1968, 718, 208–
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[5] a) A. Loudet, K. Burgess, Chem. Rev. 2007, 107, 4891–4932; b) G.
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[6] T. Gareis, C. Huber, O. S. Wolfbeis, J. Daub, Chem. Commun. 1997,
1717–1718.
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Acknowledgements
The K.U. Leuven authors are grateful to the University Research Fund
of the K.U. Leuven for grant IDO/00/001 and for postdoctoral fellow-
ships to W.Q. and M.B. The authors from the K.U. Leuven and the Uni-
versity of Wroclaw thank the Flemish Ministry of Science and Technolo-
gy for a postdoctoral fellowship to A.F. through the Bilateral Scientific
and Technological Cooperation Program (grant no. BIL05/16). Dr. A.
Stefan is acknowledged for technical help with the single-photon timing
experiments. W.Q. thanks the Scientific Research Fund for Introducing
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Received: August 9, 2010
Revised: April 27, 2011
Published online: August 17, 2011
10934
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Chem. Eur. J. 2011, 17, 10924 – 10934