BODIPY-based hydroxyaryl derivatives as fluorescent pH probes

Article abstract of DOI:10.1021/jo0503714

Seven new 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) dyes with phenolic or naphtholic subunits on position 8 and with substituents having different electron driving forces on positions 3 and 5 were synthesized. Their absorption and steady-state f

Full text of DOI:10.1021/jo0503714

BODIPY-Based Hydroxyaryl Derivatives as Fluorescent pH Probes
Mukulesh Baruah, Wenwu Qin, Nikola Basaric´, Wim M. De Borggraeve, and Noe¨l Boens*
Department of Chemistry, Katholieke Universiteit Leuven, 3001 Leuven, Belgium
noel.boens@chem.kuleuven.ac.be
Received February 28, 2005
Seven new 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) dyes with phenolic or naphtholic
subunits on position 8 and with substituents having different electron driving forces on positions
3 and 5 were synthesized. Their absorption and steady-state fluorescence properties were
investigated as a function of solvent. The novel compounds, with the exception of 4,4-difluoro-8-
(4-hydroxyphenyl)-3,5-bis-(4-methoxyphenyl)-4-bora-3a,4a-diaza-s-indacene, are characterized by
absorption maxima in the range 493-515 nm and small (400-600 cm-1) Stokes shifts. The
exceptional dye has absorption maxima in the 570-580 nm region and fluorescence emission
maxima around 610-620 nm, depending on the solvent. In aqueous solution, the dyes show a large
fluorescent enhancement upon increasing the acidity of the solution. They can be used in aqueous
solution as fluorescent pH probes excitable with visible light, with pKa values ranging from 7.5 to
9.3, depending on the substitution pattern on positions 3, 5, and 8.
Introduction
been applied as pH sensors in organic solvents or
aqueous-organic mixed media.6-8 Boradiazaindacenes
bearing phenolic,6 dialkylaminophenyl,7 and calix[4]-
Measurement of pH by fluorescence-based techniques
is well established for both imaging and sensing applica-
tions. It offers significant advantages over other tech-
niques due to its generally nondestructive character, high
sensitivity and specificity, and the wide range of indicator
dyes available.1 In recent years, a lot of work has been
focused on the synthesis2 of 4,4-difluoro-4-bora-3a,4a-
diaza-s-indacene (borondipyrromethene, BDP, or BO-
DIPY3)-based fluorescent probes and their application4
as selective and efficient sensors of ionic species. BDP
dyes have many valuable qualities such as high photo-
stability, high fluorescence quantum yields, and rela-
tively high absorption coefficients. Furthermore, they are
photoexcitable with visible light, have narrow emission
bandwidths with high peak intensities, and are amenable
to structural modification.5 BDP-based fluorophores have
(2) (a) Lee, C.-H.; Lindsey, J. S. Tetrahedron 1994, 50, 11427-11440.
(b) Wagner, R. W.; Lindsey, J. S. Pure Appl. Chem. 1996, 68, 1373-
1380. (c) Thoresen, L. H.; Kim, H.; Welch, M. B.; Burghart, A.; Burgess,
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Reibenspies, J.; Burgess, K. Chem. Commun. 1999, 1889-1890. (e)
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Burgess, K. J. Org. Chem. 1999, 64, 7813-7819. (f) Chen, J.; Burghart,
A.; Wan, C.-W.; Thai, L.; Ortiz, C.; Reibenspies, J.; Burgess, K.
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Derecskei-Kovacs, A.; Burgess, K. J. Org. Chem. 2000, 65, 2900-2906.
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(3) BODIPY is a registered trademark of Molecular Probes, Inc.,
Eugene, OR.
(4) (a) Rurack, K.; Kollmannsberger, M.; Resch-Genger, U.; Daub,
J. J. Am. Chem. Soc. 2000, 122, 968-969. (b) Rurack, K.; Kollmanns-
berger, M.; Daub, J. Angew. Chem., Int. Ed. 2001, 40, 385-387. (c)
Turfan, B.; Akkaya, E. U. Org. Lett. 2002, 4, 2857-2859. (d) Gee, K.
R.; Rukavishnikov, A.; Rothe, A. Comb. Chem. High Throughput
Screening 2003, 6, 363-366. (e) Cha, N. R.; Moon, S. Y.; Chang, S.-K.
Tetrahedron Lett. 2003, 44, 8265-8268. (f) Moon, S. Y.; Cha, N. R.;
Kim, Y. H.; Chang, S.-K. J. Org. Chem. 2004, 69, 181-183. (g) Gabe,
Y.; Urano, Y.; Kikuchi, K.; Kojima, H.; Nagano, T. J. Am. Chem. Soc.
2004, 126, 3357-3367. (h) Coskun, A.; Akkaya, E. U. Tetrahedron Lett.
2004, 45, 4947-4949.
(1) (a) Principles of Fluorescence Spectroscopy, 2nd ed.; Lakowicz,
J. R., Ed.; Kluwer Academic/Plenum Publishers: New York, 1999; pp
531-572. (b) Fluorescent Chemosensors for Ion and Molecule Recogni-
tion; Desvergne, J.-P., Czarnik, A. W., Eds.; Kluwer Academic Publish-
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Probes and Research Products, 9th ed.; Haugland, R. P., Ed.; Molecular
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Applications; Valeur, B., Ed.; Wiley-VCH: Weinheim, Germany, 2002.
10.1021/jo0503714 CCC: $30.25 © 2005 American Chemical Society
Published on Web 04/16/2005
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J. Org. Chem. 2005, 70, 4152-4157

BODIPY-Based Hydroxyaryl Derivatives
8 with neat excess pyrrole catalyzed by trifluoroacetic
acid (TFA) at room temperature. Dipyrromethane 9 was
purified, and one portion was oxidized by p-chloranil to
dipyrromethene 10. Compound 10 was separated, fol-
lowed by reaction with triethylamine (TEA) and BF3-
OEt2 in refluxing toluene to afford compound 1. The other
portion of dipyrromethane 9 was chlorinated with 2 equiv
of N-chlorosuccinimide (NCS) at -78 °C in tetrahydro-
furan (THF) according to the procedure described for the
halogenation of pyrrole10 to afford dipyrromethane 11.
For the synthesis of dye 2 (Scheme 2), we first prepared
5-(4-hydroxyphenyl)-1,9-dimethyl dipyrromethene 13
through a known procedure2 by condensing aldehyde 8
with 2 equiv of 2-methylpyrrole (12) in CH2Cl2 at room
temperature in the presence of TFA as catalyst, followed
by oxidation with p-chloranil. Product 13 was further
reacted with TEA and BF3-OEt2 to afford compound 2.
The BDP derivatives 5, 6, and 7 were also synthesized
by the same procedure2 by condensing 2-methylpyrrole
(12)11 with 3-chloro-4-hydroxybenzaldehyde, 3-hydroxy-
benzaldehyde, and 6-hydroxy-2-naphthaldehyde,12 re-
spectively. Compound 4 was synthesized similarly2 from
aldehyde 8 and 2-(4-methoxyphenyl) pyrrole.2c
FIGURE 1. BODIPY-based pH probes.
arene8 subunits at position 8 all showed deprotonation/
protonation dependent fluorescence off/on switching. The
phenolic, N,N-dialkylaniline, and calix[4]arene deriva-
tives sense the alkaline, the acidic, and the near neutral
pH range, respectively. The low emission intensity of the
phenolate6,8 or uncharged dialkylaniline7 forms has been
attributed to charge transfer from the phenolate or
uncharged N,N-dialkylaniline donors to the excited-state
indacene acceptor moiety.
The present study is directed toward the synthesis and
fluorimetric characterization of BDP fluorophores that
can probe pH changes by large associated changes in the
emission intensity around 510 nm and possess pKa values
close to the physiological pH range. Seven 4,4-difluoro-
4-bora-3a,4a-diaza-s-indacene dyes with phenolic or naph-
tholic subunits at position 8 have been investigated
(compounds 1-7 in Figure 1). The BDP compounds have
no substituents at the 1 and 7 positions, whereas the 3
and 5 positions are substituted with groups having
different electron driving forces to study the effect on pKa
and fluorescence quantum yields.
The purified and characterized (1H, 13C NMR, mass
spectra) new BDP compounds were dissolved in several
solvents to study their spectroscopic properties (Table 1).
The shortest wavelength absorption (or excitation) and
emission were observed for the 3,5-unsubstituted com-
pound 1, which also had the lowest fluorescence quantum
yields φf. The 3,5-dimethylBDP compounds 2, 5, 6, and
7 all have their maxima of excitation (505-514 nm) and
emission (517-530 nm) within a very narrow wavelength
range in the four solvents. The influence of the 8-sub-
stituent on the absorption and emission maxima of the
3,5-dimethylBDP probes is minimal, even for the 8-naph-
thol derivative 7. 3,5-DichloroBDP compound 3 has
excitation (506-515 nm) and emission (517-527 nm)
maxima similar to those of the 3,5-dimethylBDP com-
pounds 2, 5, 6, and 7. As compared to the other probes,
compound 4 with p-methoxyphenyl substituents in posi-
tions 3 and 5 shows a clear shift in the excitation (570-
581 nm) and emission (612-618 nm) maxima toward
longer wavelength, along with an increase of φf. This is
due to the increasing conjugation of the π-system of the
chromophore. While the Stokes shifts for all other
compounds are small (∆νj ) 370-650 cm-1), derivative 4
has relatively large Stokes shifts (1030-1380 cm-1).
Compound 4 is not soluble in water, so further study in
aqueous solution is not feasible. For all compounds, the
excitation and emission maxima shift to the red wave-
length region with decreasing solvent polarity, indicating
the more polar character of the ground state. The most
bathochromic excitation and emission is found in toluene
as solvent. Concurrently, the fluorescence quantum yields
decrease with increasing solvent polarity. The highest φf
values are measured in toluene solution, the second
Results and Discussion
We synthesized compounds that are 3,5-unsubstituted
(1), 3,5-disubstituted with methyl (2, 5, 6, and 7) and
p-methoxyphenyl (4) groups (electron-donating function-
alities), and 3,5-disubstituted with chlorine atoms (3)
(inductively electron-withdrawing group). The syntheses
of 1 and 3 (Scheme 1) were carried out through an
existing procedure2 starting from 4-hydroxybenzaldehyde
(8). The needed dipyrromethane 9 was prepared accord-
ing to a published procedure9 by condensation of aldehyde
(5) (a) Treibs, A.; Kreuzer, F.-H. Liebigs Ann. Chem. 1968, 718, 208-
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Am. Chem. Soc. 1994, 116, 7801-7806.
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D. F.; Boyle, P. D.; Lindsey, J. S. J. Org. Chem. 1999, 64, 1391-1396.
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2225.
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Stewart, A. O.; Bouska, J. B.; Harris, R. R.; Hulkower, K. I.; Malo, P.
E.; Bell, R. L.; Carter, G. W.; Brooks, C. D. W. J. Med. Chem. 2000,
43, 690-705.
(7) (a) Kollmannsberger, M.; Gareis, T.; Heinl, S.; Breu, J.; Daub,
J. Angew. Chem., Int. Ed. Engl. 1997, 36, 1333-1335. (b) Werner, T.;
Huber, C.; Heinl, S.; Kollmannsberger, M.; Daub, J.; Wolfbeis, O. S.
Fresenius’ J. Anal. Chem. 1997, 359, 150-154. (c) Kollmannsberger,
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J. Org. Chem, Vol. 70, No. 10, 2005 4153

Baruah et al.
SCHEME 1. Synthesis of 1 and 3
SCHEME 2. Synthesis of 2
TABLE 1. Fluorescence Characteristics of the BDP
Dyes in Different Solvents
of the synthesized BDP derivatives are high and lie in
the 60 000-110 000 M-1 cm-1 range.
The acidity constants Ka of probes 1-3 and 5-7 were
determined in aqueous nonbuffered solution by fluori-
metric titration as a function of pH using the fluorescence
excitation and/or emission spectra. Nonlinear curve-
fitting of eq 1 to the fluorescence data F recorded as a
function of pH yielded values of Ka, the fluorescence
signals Fmin and Fmax at minimal and maximal [H+],
respectively, and n (the stoichiometry of H+ binding to
the phenolate or naphtholate form of the probe). Fitting
eq 1 to the steady-state fluorescence data F with n, Ka,
Fmin, and Fmax as freely adjustable parameters always
gave values of n close to 1, indicating that one proton is
bound per phenoxide or naphthoxide anion. Therefore,
n was kept fixed at 1 in the final curve fittings, from
which the estimated values of Ka, Fmin, and Fmax are
reported here.
λAbs
λem
λex
∆maxυ
BDP
1
solvent
(max/nm) (max/nm) (max/nm) (cm-1
)
φf
MeOH
MeCN
493
493
499
501
505
505
510
511
506
506
513
515
570
567
581
507
507
513
515
508
507
512
514
508
507
512
514
508
508
512
518
517
517
521
524
517
518
523
527
612
615
618
520
521
526
529
521
521
525
528
522
524
527
530
494
493
499
501
505
505
510
511
506
506
513
515
570
567
581
507
508
513
514
507
507
512
513
508
507
512
513
599 0.015
599 0.023
509 0.043
655 0.081
460 0.28
496 0.24
414 0.45
486 0.63
420 0.014
458 0.089
373 0.33
442 0.63
1204 0.57
1377 0.48
1031 0.68
493 0.16
530 0.20
482 0.28
514 0.56
491 0.19
530 0.17
484 0.25
516 0.44
528 0.025
640 0.058
556 0.17
587 0.26
cyclohexane
toluene
MeOH
2
3
MeCN
cyclohexane
toluene
MeOH
MeCN
cyclohexane
toluene
MeOH
4
5
MeCN
toluene
MeOH
MeCN
cyclohexane
toluene
MeOH
Fmax[H+]n + FminKa
F )
(1)
6
7
Ka + [H+]n
MeCN
cyclohexane
toluene
MeOH
As examples, the absorption, emission, and excitation
spectra of 5 are shown in Figure 2 as a function of pH.
The absorption spectra (Figure 2a) show a small shift of
the maximum (from 505 to 502 nm) with increasing pH
and a clear isosbestic point around 392 nm. The fluores-
cence emission (Figure 2b) and excitation (Figure 2c)
intensities increase at lower pH values. There is no
significant shift of the absorption (or excitation) and
emission maxima as a function of pH (Table 2). Figure 3
is a representative example of the fit of eq 1 with n ) 1
to the fluorescence emission data of 5 as a function of
[H+].
MeCN
cyclohexane
toluene
highest in cyclohexane. The position of the hydroxyl and
3,5-dimethylBDP functionalities on the benzene ring in
dyes 2 (para) and 6 (meta) has no significant influence
on the spectral properties and fluorescence quantum
yields. 3,5-DichloroBDP compound 3 shows the largest
increase of φf when going from protic polar methanol to
apolar toluene. The molar absorption coefficients for most
4154 J. Org. Chem., Vol. 70, No. 10, 2005

BODIPY-Based Hydroxyaryl Derivatives
FIGURE 3. The solid line represents the best fit of eq 1 with
n ) 1 to the fluorimetric titration data of dye 5 obtained from
the emission spectra of Figure 2b (λex ) 488 nm, λem ) 520
nm).
group is 8.75. In contrast, the 3,5-dichloroBDP substitu-
ent (in compound 3) decreases the pKa to 8.41.
These observed effects are in line with the electron-
withdrawing (or releasing) power of the substituents in
R-position to the nitrogen atoms (Table 2). When phenol
is substituted in the meta position with 3,5-dimethylBDP
(in compound 6), the pKa is 9.34. When o-chlorophenol
(with a pKa of 8.53 13) is substituted at the para position
with 3,5-dimethylBDP as in dye 5, the pKa is reduced to
7.49. In contrast to the phenol derivatives, the pKa of
2-naphthol (∼9.5)14,15 is hardly affected by the boradi-
azaindacene moiety in compound 7. Also, the fluorescence
excitation and emission spectra of compound 7 are those
of 3,5-dimethylBDP. Probe 5, where a chlorine atom is
introduced in the ortho position to the OH functionality
in the phenyl ring, has the most favorable pKa value for
intracellular pH measurements. The high photostability,
the capability of using visible light excitation (necessary
to avoid autofluorescence of most biological samples), the
pKa suitable for near-neutral pH measurements, the
solubility in aqueous solution, the negligible sensitivity
of pKa to low ionic strength (see below), the high molar
absorption coefficients, and the high fluorescence en-
hancement factor Fmax/Fmin make molecule 5 an interest-
ing lead compound for designing biologically useful
fluorescent off/on pH probes.
To study the influence of buffer and added salt on the
steady-state fluorescence, we recorded pH-dependent
fluorescence emission and excitation spectra of dye 5
under various conditions. First, the steady-state fluores-
cence spectra of compound 5 were measured in the
presence of different concentrations of MOPS (3-[N-
morpholino]propanesulfonic acid) buffer but in the ab-
sence of added salt. Second, measurements in the absence
and presence of various concentrations of MOPS buffer
were performed in the presence of 0.1 M KCl. The pKa
values obtained are compiled in Table 3, indicating that
FIGURE 2. (a) Absorption spectra of compound 5 in aqueous
nonbuffered solution as a function of pH. (b) Corresponding
fluorescence emission spectra (λex ) 488 nm) and (c) fluores-
cence excitation spectra (λem ) 560 nm).
The pKa of the phenol derivatives 1, 2, 3, 5, and 6 can
be tuned by varying the substituents on BDP. When
phenol (with a pKa of 10.00)13 is substituted at the para
position with the BDP moiety (compound 1), the pKa
drops to 8.69. When the R-positions to the nitrogen atoms
are substituted with the (inductively) electron-donating
methyl group (in compound 2), the pKa of the phenolic
(14) Ellis, D. W. J. Chem. Educ. 1966, 43, 259-261.
(15) Van den Bergh, V.; Boens, N.; De Schryver, F. C.; Ameloot, M.;
Gallay, J.; Kowalczyk, A. Chem. Phys. 1992, 166, 249-258.
(13) Physical Organic Chemistry, 2nd ed.; Isaacs, N. S., Ed.; Long-
man Scientific & Technical: Harlow, England, 1995; p 188.
J. Org. Chem, Vol. 70, No. 10, 2005 4155

Baruah et al.
TABLE 2. Photophysical Properties of the BDP Derivatives in Aqueous Nonbuffered Solutiona
λabs-acid/λabs-base
λem-acid/λem-base
BDP
(max/nm)
(max/nm)
φf-acid (pH)
φf-base (pH)
pKa
Fmax/Fmin
1
2
3
5
6
7
493/489
505/501
508/503
505/502
507/506
506/506
510/510
510/509
521/523
521/519
523/523
525/526
0.017 (7.10)
0.12 (7.15)
0.008 (6.50)
0.09 (5.30)
0.11 (7.45)
0.02 (6.56)
0.0007 (10.12)
0.007 (9.95)
0.0008 (10.00)
0.0007 (10.48)
0.009 (10.55)
0.003 (10.98)
8.69 ( 0.02
8.75 ( 0.05
8.41 ( 0.03
7.49 ( 0.03
9.34 ( 0.04
9.20 ( 0.03
40
25
8
130
35
6
a Ka, Fmax, and Fmin values were determined by fitting eq 1 with n ) 1 to the fluorescence data. The fluorescence quantum yields φf-base
and φf-acid were determined at, respectively, the highest and lowest pH values (between parentheses) used in the fluorimetric titration.
TABLE 3. pKa Values of Dye 5 Determined by Direct
Fluorimetric Titration in the Absence/Presence of MOPS
Buffer and Added Salt (KCl)a
distillation. The residue was chromatographed on silica, start-
ing elution with CH2Cl2 and gradually increasing the polarity
by adding EtOAc (for 1, up to 100% (v) EtOAc; for 3, up to
50% EtOAc; for 4, up to 5% EtOAc).
C (M)
ionic strength (M)
pKa
4,4-Difluoro-8-(4-hydroxyphenyl)-4-bora-3a,4a-diaza-
s-indacene (1). Yield: 64%. Orange crystals mp 153 °C;
recrystallized from chloroform/cyclohexane. 1H NMR (CD-
Cl3): δ 6.15 (broad s, 1H, OH), 6.56 (m, 2H), 6.97 (d, 2H, J )
8.8 Hz), 6.99 (m, 2H), 7.47 (d, 2H, J ) 8.8 Hz), 7.93 (m, 2H).
13C NMR (CDCl3): δ 116.0 (d), 118.8 (d), 126.6 (s), 131.9 (d),
133.0 (d), 135.2 (s), 143.8 (d), 147.9 (s), 159.1 (s). LRMS (EI,
70 eV): m/z (%) 285 (M+, 20), 284 (M+, 100), 283 (M+, 80), 263
(20), 198 (30), 155 (50), 121 (40), 91 (100). HRMS calcd. M+
for C15H11BF2N2O 284.09325, found 284.09342.
4,4-Difluoro-8-(4-hydroxyphenyl)-3,5-dichloro-4-bora-
3a,4a-diaza-s-indacene (3). Yield: 47%. Red crystals mp 224
°C; recrystallized from chloroform/cyclohexane. 1H NMR
(CDCl3): δ 5.77 (broad s, 1H, OH), 6.45 (d, 2H, J ) 4.4 Hz),
6.89 (d, 2H, J ) 4.4 Hz), 6.99 (d, 2H, J ) 8.8 Hz), 7.42 (d, 2H,
J ) 8.8 Hz). 13C NMR (CDCl3): δ 116.3 (d), 119.0 (d), 127.7
(s), 132.0 (d), 132.9 (d), 134.0 (s), 144.3 (s), 159.6 (s), 172.7 (s).
LRMS (EI, 70 eV): m/z (%) 354 (M+, 5), 353 (M+, 5), 352 (M+,
15), 317 (15), 304 (15), 269 (40), 232 (40), 198 (35), 155 (60),
121 (40), 91 (100). HRMS calcd. M+ for C15H9BCl2F2N2O
352.01530, found 352.01521.
0 M MOPS + 0 M KCl
0
7.49 ( 0.03
7.45 ( 0.03
7.41 ( 0.05
7.48 ( 0.03
7.35 ( 0.05
0.01 M MOPS + 0 M KCl
0.05 M MOPS + 0 M KCl
0 M MOPS + 0.1 M KCl
0.05 M MOPS + 0.1 M KCl
0.02
0.10
0.10
0.20
a The values of the ground-state acidity constants Ka measured
from emission spectra (λex ) 488 nm and λem ) 520, 530 nm) and
excitation spectra (λem ) 560 nm and λex ) 506, 516 nm) are in
excellent agreement. The ionic strength values were calculated
for solutions at 20 °C characterized by pH 7.20 (equal to the pKa
of MOPS).
the ground-state acidity constants of 5 are nearly insen-
sitive to added salt and/or MOPS buffer in the concentra-
tion range studied. As the effects of added salt and buffer
are very small, pH probe 5 has the additional advantage
of a negligible sensitivity to low ionic strength.
Conclusion
4,4-Difluoro-8-(4-hydroxyphenyl)-3,5-bis-(4-meth-
oxyphenyl)-4-bora-3a,4a-diaza-s-indacene (4). Yield: 30%.
Deep blue crystals; mp 250 °C; recrystallized from chloroform/
cyclohexane. 1H NMR (CDCl3): δ 3.86 (s, 6H, OCH3), 6.62 (d,
2H, J ) 4.4 Hz), 6.89 (d, 2H, J ) 4.4 Hz), 6.97 (d, 4H, J ) 8.8
Hz), 6.99 (d, 2H, J ) 8.8 Hz), 7.49 (d, 2H, J ) 8.8 Hz), 7.88 (d,
4H, J ) 8.8 Hz), OH is not seen. 13C NMR (CDCl3): δ 55.7 (q),
114.2 (d), 115.7 (d), 120.7 (d), 125.7 (s), 127.5 (s), 130.7 (d),
131.4 (d), 131.59 (s), 131.55 (s), 132.8 (d), 151.9 (s), 157.9 (s),
161.0 (s). LRMS (EI, 70 eV): m/z (%) 497 (M+, 30), 496 (M+,
100), 495 (M+, 20), 481 (35), 480 (5), 206 (50), 170 (40). HRMS
calcd. M+ for C29H23BF2N2O3 496.17698, found 496.17685.
Synthesis of 4,4-Difluoro-3,5-dimethyl-8-(4-hydroxy-
phenyl)-4-bora-3a,4a-diaza-s-indacene (2). 100 mg (0.38
mmol) of compound 13 was dissolved in 60 mL of absolute THF
purged with argon. To the solution was added 1.5 mL of
absolute triethylamine, and the mixture was stirred for 0.5 h
at room temperature under argon atmosphere. 1.5 mL of BF3-
OEt2 was added dropwise through a syringe, and the mixture
was stirred for 6-7 h (sometimes overnight) at room temper-
ature. A deep green fluorescence was observed in the mixture.
The solvent was evaporated, and the crude product was
purified by column chromatography over silica gel with pure
ethyl acetate to afford a red powder, which was recrystallized
from hexane/chloroform to yield 37 mg (31%) of red crystals,
mp 156-157 °C. 1H NMR (CDCl3): δ 2.66 (s, 6H), 6.27 (d, 2H,
J ) 4.3 Hz), 6.74 (d, 2H, J ) 4.3 Hz), 6.91 (d, 2H, J ) 8.1 Hz),
7.38 (d, 2H, J ) 8.1 Hz). 13C NMR (CDCl3): δ 15.3, 115.6,
119.6, 130.7, 132.6, 134.9, 143.0, 157.4, 158.0, 167.4. LRMS
(EI, 70 eV): m/z (%) 312 (M+ 100); 311 (M+ 61), 292(67), 291
(60). HRMS calcd. M+ for C17H15BF2N2O 312.12455, found
312.12423.
We synthesized seven new 4,4-difluoro-4-bora-3a,4a-
diaza-s-indacene dyes (1-7) with phenolic or naphtholic
subunits (Figure 1). Their absorption and steady-state
fluorescence properties were investigated as a function
of solvent. Compounds 1-3 and 5-7 are characterized
by absorption maxima in the range 493-515 nm and
small (400-600 cm-1) Stokes shifts. Fluorescent dye 4
has absorption maxima in the 570-580 nm region and
fluorescence emission maxima around 610-620 nm,
depending on the solvent. In aqueous solution, com-
pounds 1-3 and 5-7 show a large fluorescent enhance-
ment upon increasing the acidity of the solution. They
can be used in aqueous solution as fluorescent pH probes
excitable with visible light, with pKa values ranging from
7.5 to 9.3, depending on the substitution pattern on
positions 3, 5, and 8.
Experimental Section
General Procedure for the Preparation of Phenolic
BODIPYs 1, 3, and 4. A toluene solution of 1 equiv of
dipyrromethene was purged with argon. To the solution was
added 10 equiv of triethylamine. This solution was heated at
70 °C for 0.5 h. Next, 15 equiv of BF3-OEt2 was added and
the reaction was heated at reflux temperature for 2 h. To the
cooled reaction mixture was added a 1 M aqueous solution of
sodium hydroxide. The layers were separated. The water layer
was brought to pH ∼5-6 by addition of HCl and extracted
with dichloromethane. The combined organic layers were dried
over magnesium sulfate, and the solvent was removed by
Indicators 5, 6, and 7 were prepared by the same procedure
as for 2.
4156 J. Org. Chem., Vol. 70, No. 10, 2005

BODIPY-Based Hydroxyaryl Derivatives
4,4-Difluoro-3,5-dimethyl-8-(3-chloro-4-hydroxyphenyl)-
4-bora-3a,4a-diaza-s-indacene (5). Yield 42%. Red crystals;
mp 180-181 °C; recrystallized from chloroform/cyclohexane.
1H NMR (CDCl3): δ 2.66 (s, 6H), 6.29 (d, 2H, J ) 4.4 Hz),
6.73 (d, 2H, J ) 4.4 Hz), 7.12 (d, 1H, J ) 8.7 Hz), 7.35 (dd,
1H, J ) 8.0 Hz, J ) 2.2 Hz), 7.51 (d, 1H, J ) 2.2 Hz). 13C
NMR (CDCl3): δ 15.3, 27.3, 116.5, 119.9, 120.4, 127.9, 130.5,
131.2, 131.2, 134.7, 153.4, 158.2. LRMS (EI, 70 eV): m/z (%)
348 (M+ 68), 346 (M+ 100), 327 (54), 326 (97), 325 (91). HRMS
calcd. M+ for C17H14BClF2N2O 346.08558, found 346.08510.
4,4-Difluoro-3,5-dimethyl-8-(3-hydroxyphenyl)-4-bora-
3a,4a-diaza-s-indacene (6). Yield 52%. Shiny green crystals;
only dilute solutions with an absorbance below 0.1 at the
excitation wavelength λex were used. Rhodamin 6G in H2O (λex
) 488 nm, φf ) 0.76) and/or Cresyl Violet (λex ) 546 nm, φf )
0.55) in methanol were used as fluorescence standard.16 The
quantum efficiencies of fluorescence in this work were obtained
from average values of multiple (generally four) independent
measurements. Correction for the refractive index was applied.
All spectra were recorded at 20 °C.
Determination of Ka via Fluorimetric Titration. To
determine the acidity constants Ka of 1-3 and 5-7, these
probes were dissolved in 1.0 M NaOH solution. Next, 0.1 and
0.01 M solutions of HCl and/or NaOH were used to adjust the
pH of the solutions. All of the measurements were done at 20
°C on nonbuffered solutions, unless specified otherwise.
Determination of Ka of Compound 5 in the Presence/
Absence of MOPS Buffer/KCl. An appropriate amount of
dye 5 in 1.0 M NaOH solution (with a final absorbance below
0.1 at the excitation wavelength λex after dilution), 0 or 2 or
10 mL of 0.1 M MOPS, and 0 or 1 mL of 2.0 M KCl were
pipetted into a 20 mL measuring flask. The mixture was
diluted to 20 mL with Milli-Q water. A few microliters of 0.1
and 0.01 M solutions of HCl and/or NaOH were used to adjust
the pH of the solutions. All of the measurements were done
at 20 °C.
1
mp > 300 °C recrystallized from chloroform/cyclohexane. H
NMR (CDCl3): δ 2.66 (s, 6H), 6.26 (d, 2H, J ) 4.4 Hz), 6.75
(d, 2H, J ) 4.4 Hz), 6.97-7.07 (m, 3H), 7.32 (m, 1H). 13C NMR
(CDCl3): δ 15.3, 117.3, 117.7, 119.8, 123.5, 129.9, 130.8, 134.8,
135.9, 155.6, 158.1. LRMS (EI, 70 eV): m/z (%) 312 (M+ 100),
311 (M+ 60), 292 (68), 291 (63). HRMS calcd. M+ for C17H15-
BF2N2O 312.12455, found 312.12328.
4,4-Difluoro-3,5-dimethyl-8-(6-hydroxynaphth-2-yl)-4-
bora-3a,4a-diaza-s-indacene (7). Yield 31%. Red crystals;
mp 223-224 °C; recrystallized from chloroform/cyclohexane.
1H NMR (CDCl3): δ 2.68 (s, 6H), 6.28 (d, 2H, J ) 4.1 Hz),
6.76 (d, 2H, J ) 4.1 Hz), 7.18-7.24 (m, 2H), 7.55 (dd, 1H, J )
8.4 Hz, J ) 1.8 Hz), 7.75-7.84 (m, 2H), 7.92 (s, 1H). 13C NMR
(CDCl3): δ 15.3, 47.2, 109.8, 119.3, 119.7, 126.7, 128.4, 128.8,
129.8, 130.7, 130.9, 135.1, 135.6, 155.2, 157.7. LRMS (EI, 70
eV): m/z (%) 363 (M+ 20), 362 (M+ 100), 361 (M+ 45), 342 (45),
341 (30). HRMS calcd. M+ for C21H17BF2N2O 362.14020, found
362.14059.
Materials. All solvents were spectroscopic grade and were
used without further purification. Potassium chloride (KCl,
99.999%) and MOPS (3-[N-morpholino]propanesulfonic acid,
>99.5%) were used as such. The chemicals used for the
synthesis were of the best grade available and were used as
received. Dichloromethane (p.a.) was dried over molecular
sieves. Boron trifluoride etherate (BF3-OEt2) was ca. 48% BF3.
Steady-State Spectroscopy. The absorption measure-
ments were performed on a Perkin-Elmer Lambda 40 UV/vis
spectrophotometer. Corrected steady-state excitation and emis-
sion spectra were recorded on a SPEX Fluorolog. For the
determination of the relative fluorescence quantum yields (φf),
Acknowledgment. We thank the University Re-
search Fund of the K.U. Leuven for grant IDO/00/001
and for postdoctoral fellowships to M.B., N.B., and W.Q.
W.M.D.B. is a postdoctoral researcher of the Fonds voor
Wetenschappelijk Onderzoek - Vlaanderen (FWO).
Supporting Information Available: All experimental
procedures and spectroscopic characterization (1H and 13C
NMR, mass spectra) of compounds 1-7 and some inter-
mediates; fluorescence excitation and emission spectra. This
material is available free of charge via the Internet at
http://pubs.acs.org.
JO0503714
(16) Olmsted, J. J. Phys. Chem. 1979, 83, 2581-2584.
J. Org. Chem, Vol. 70, No. 10, 2005 4157