Preparation of BODIPY probes for multicolor fluorescence imaging studies of membrane dynamics

Article abstract of DOI:10.1039/b100757m

Three different colored fluorescent fatty acids containing BODIPY with extremely high fluorescence quantum yields have been synthesized as probes for investigating the dynamics of membranes. Colored vesicles containing each probe, which were located in th

Full text of DOI:10.1039/b100757m

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Preparation of BODIPY probes for multicolor Ñuorescence imaging
studies of membrane dynamics
Koji Yamada, Taro Toyota, Katsuto Takakura, Masako Ishimaru and Tadashi Sugawara*
Department of Basic Science, Graduate School of Arts and Sciences, T he University of T okyo,
3-8-1 Komaba, Meguro, T okyo 153-8902, Japan. E-mail: suga=pentacle.c.u-tokyo.ac.jp
L e t t e r
Received (in Montpellier, France) 22nd January 2001, Accepted 28th February 2001
First published as an Advance Article on the web 11th April 2001
Three di†erent colored Ñuorescent fatty acids containing
BODIPY with extremely high Ñuorescence quantum yields
have been synthesized as probes for investigating the dynamics
of membranes. Colored vesicles containing each probe, which
were located in the interior of the bilayer membranes, were dis-
tinguished from each other by Ñuorescence microscopy.
difficulties. In this work we report the preparation of highly
Ñuorescent diaryl derivatives of BODIPY carrying methyl
groups at the ortho positions (R ) of the phenyl group substi-
2
tuted at C(8) of the s-indacene skeleton.
4-Hydroxybenzaldehyde
and
2,6-dimethyl-4-hydroxy-
benzaldehyde, the latter obtained from 3,5-dimethylphenol
using the ReimerÈTiemann reaction,7 were reacted with ethyl
11-bromoundecanoate to a†ord a fatty acid ester. The formyl
group of the product can be converted to the pigment by the
following treatment.8 Condensation of the benzaldehyde
derivatives with the corresponding substituted pyrroles, which
were synthesized in the same manner as in previous
papers,9,10 followed by an oxidation procedure gave dipyrro-
methane intermediates, which were treated with BF É Et O to
Analysis of the dynamics of membranes is important for
understanding biological functions in living cells.1 Fluores-
cence microscopic analysis is of use for this purpose because
the dynamic behavior of membranes can be visualized
directly.2 Nowadays hundreds of Ñuorescence probes are com-
mercially available and they have been used in various bio-
logical experiments.3 Among Ñuorophores, 4,4-diÑuoro-4-
bora-3a,4a-diaza-s-indacene (BODIPY) is lipophilic and
exhibits high Ñuorescence efficiency and excellent photo-
stability.4 The advantage of BODIPY derivatives is that their
absorption and emission maxima can be shifted to longer
wavelengths by chemical modiÐcation of the p-conjugating
system.5 Accordingly, BODIPY derivatives may be suitable
for investigating mixed membranes composed of di†erent
membrane components, which are required to be distin-
guished from each other simultaneously. In this communica-
tion, we report the synthesis of three spectroscopically
di†erentiable Ñuorescent probes 2c, 4b and 6b from readily
available chemicals. The problem with BODIPY derivatives
carrying aryl groups at the 3- and 5-positions of the s-
indacene skeleton is the low quantum yield of Ñuorescence.5
As for alkyl homologs, the Ñuorescence quantum yield can be
enhanced by introducing methyl groups at the C(1) and C(7)
3
2
produced Ñuorescent esters 1, 3 and 5.5 Finally, a convention-
al alkaline hydrolysis gave the desired fatty acid probes 2, 4
and 6 (Scheme 1).
The Ñuorescence of the probes turned out to be diminished
in non-polar solvents. This might be because 2, 4 and 6 are
thought to form a hydrogen-bonded dimer in these solvents
and their Ñuorescence is self-quenched. This interpretation is
supported by the fact that the Ñuorescence intensity was
partly restored when a small amount of acetic acid was added
to the solution. In order to estimate their photo-physical
properties, the absorption and emission spectra of ethyl esters
1, 3 and 5 were measured in dichloromethane (Fig. 1). The
absorption spectra of 1aÈd, 3a,b and 5a,b show absorption
maxima (j ) in the ranges 500È511, 548È553 and 572È578
abs
nm, respectively. These maximum positions are mainly deter-
mined by the substituent at the C(3) and C(5) positions,
although they vary slightly depending on the nature (Me or
H) of the R and/or R substituents. The Ñuorescence spectra
positions as R .5,6 However, introduction of methyl groups at
3
these positions of diaryl derivatives is not without its synthetic
2
3
DOI: 10.1039/b100757m
New J. Chem., 2001, 25, 667È669
667
This journal is ( The Royal Society of Chemistry and the Centre National de la Recherche ScientiÐque 2001

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Table
1
Photophysical properties of BODIPY compounds in
CH Cl
2
2
Compound
j
/nm
j
a/nm
Stokes shift/nm
U b
abs
Em
f
1a
1b
1c
1d
3a
3b
5a
5b
508
511
500
501
548
553
572
578
524
524
516
512
583
587
616
620
16
13
16
13
35
34
44
42
0.36c
0.88
0.71c
0.94
0.21c
0.68
0.30c
0.56
a Excited at j . b N,N@-Bis(1-hexylheptyl)-3,4 : 9,10-perylenebis(di-
abs
carboximide) was used as a reference (U \ 1.00). c Similar Ñuoro-
f
Scheme 1 (i) Ethyl 11-bromoundecanoate, K CO , acetone; (ii) 2-
phores in which a p-iodophenyl group had been introduced at C(8) of
2
3
substituted pyrrole, CF COOH, CH Cl ; (iii) DDQ, CH Cl ; (iv)
the BODIPY derivatives exhibited lower Ñuorescence quantum yields
compared with the current BODIPY derivatives (1a, 1c, 3a and 5a).
The suppression of the quantum yields may be ascribed to acceler-
ation of intersystem crossing from S to T by the heavy atom e†ect.
3
2
2
2 2
BF É Et O, Et N, toluene, reÑux; (v) KOH, ethanol, reÑux.
3
2
3
of 1aÈd, 3a, b and 5a,b were found to show emission maxima
1
1
(j ) in the ranges 512È524, 583È587 and 616È620 nm, respec-
Em
tively, suggesting that their Ñuorescent colors are determined
by the substituent at the C(3) and C(5) positions as well: 1aÈd
are green, 3a,b are orange and 5a,b are wine-red. These spec-
tral features facilitate signal identiÐcation and analysis when
these probes are buried in membranes.
Fluorescence quantum yields for 1, 3 and 5 were meas-
uredusingN,N@-bis(1-hexylheptyl)-3,4:9,10-perylenebis(carbox-
imide) as a reference compound.11 The quantum yields of the
Ñuorophores having methyl groups at the ortho positions of
In summary, we have revealed that introduction of methyl
groups at the ortho positions of aryl-substituted BODIPYs is
an efficient way to increase the Ñuorescence quantum yield.13
Three fatty acid probes tagged with an individual Ñuorophore
were buried within oleic acid vesicles and these vesicles were
identiÐed by multicolor Ñuorescence microscopy at the same
time. Moreover, these Ñuorescent probes, having a carboxyl
group at the terminus, can be attached easily to lysophospho-
lipids. Investigations on the dynamics of mixed membranes
composed of multi-amphiphiles and multi-probes are in
progress in our laboratories.
the phenyl group (R ) and/or at the C(1) and C(7) positions
2
(R ), are two or three times larger than those of the derivatives
3
without methyl groups at these positions (Table 1). This trend
indicates that the methyl groups suppress non-radiative deac-
tivation by restricting internal rotation of the phenyl ring at
the C(8) position. As for the synthesis of 1d, the synthetic yield
of the corresponding dipyrromethane is low due to the steric
hindrance induced by the methyl groups at R and R . On
Experimental
Syntheses
2
3
the other hand, 2-methylpyrrole, which is the starting material
for 1b, is not commercially available. Considering the above
situation, 1c is the most suitable compound for a large scale
synthesis.
2,6-Dimethyl-4-hydroxybenzaldehyde. 3,5-Dimethylphenol
(92.97 g, 0.76 mol) and KOH (78.00 g, 1.39 mol) were dis-
solved in water (300 ml). Distilled CHCl (121 ml, 0.68 mol)
3
was added over 10 h, keeping the temperature at 60 ¡C, and
When Ñuorescent images of vesicles composed of oleic acid
containing several mol% of fatty acid probes 2c, 4b and
6b were recorded, only membranes were found to glisten
strongly. Since the pigments are highly lipophilic, they are
considered to be buried di†usely in the interior of the bilayer
membrane, no appreciable self-quenching being observed.12
Furthermore, the colored vesicles containing each Ñuorescent
probe turned out to be clearly distinguishable from each
other, even when using an identical optical Ðlter (Fig. 2).
Accordingly, this technique enables the video recording of the
dynamic behavior of membranes.
the solution was stirred at 60 ¡C for 8 h. The solution was
cooled and then poured into dilute H SO . The yellow
2
4
residue was washed with CHCl to yield 10.09 g (10%) of
3
grayish white solid. 1H NMR (270 MHz, DMSO-d ): d 10.29
6
(1H, s), 10.28 (1H, br s), 6.50 (2H, s), 3.41 (6H, s). 13C NMR
(67.5 MHz, DMSO-d ): d 191.1, 161.2, 144.0, 124.1, 116.1,
6
20.6.
General procedure for the synthesis of 1, 3 and 5. A mixture
of 4-hydroxybenzaldehyde derivative (with or without methyl
groups at the 2 and 6-positions) and ethyl 11-undecanoate
was reÑuxed in dry acetone overnight in the presence of pot-
assium carbonate. The crude mixture was puriÐed by silica gel
column chromatography using CHCl to a†ord an ester-
3
tagged benzaldehyde (56È83%). The obtained benzaldehyde
(1.0 mmol) and the corresponding substituted pyrrole (2.0
mmol) were dissolved in CH Cl (25 ml) and the solution was
2
2
degassed by bubbling nitrogen through it (30 min). One drop
of CF COOH was added and the solution was stirred over-
3
Fig. 1 Normalized absorption and Ñuorescence spectra of (ÈÈ) 1c,
(È È) 3b and (È È È) 5b in CH Cl .
Fig. 2 Fluorescent microscopic images of oleic acid vesicles contain-
ing 2c (left), 4b (center) and 6b (right).
2
2
668
New J. Chem., 2001, 25, 667È669

View Article Online
night at room temperature under a nitrogen atmosphere. 2,3-
dichloro-5,6-dicyano-1,4-benzoquinone (0.25 g, 1.1 mmol) was
added, and the mixture was stirred for another 4 h. The reac-
6b. Yield 97%. IR (KBr): l/cm~1 1705 (C2O). 1H NMR (270
MHz, pyridine-d ): d 8.14 (4H, d, J \ 9), 7.19 (4H, d, J \ 9),
5
7.03 (2H, d, J \ 4), 6.98 (2H, s), 6.69 (2H, d, J \ 4), 4.09 (2H, t,
tion mixture was washed with saturated aqueous NaHCO
J \ 7), 3.70 (6H, s), 2.53 (2H, t, J \ 7), 2.20 (6H, s), 1.9È1.7
(4H, m), 1.5È1.2 (12H, m).
3
and brine, dried over Na SO , Ðltered, and concentrated.
2
4
The crude product was puriÐed by alumina column chro-
matography using hexaneÈCH Cl to a†ord the dipyrrome-
2
2
Recording of images
thane derivative as a colored oil. The product (0.26È0.41 g,
0.44È0.60 mmol) and BF É Et O (0.72 ml, 3.8 mmol) were dis-
The Ñuorescent images were recorded using an Olympus
Power BX51 microscope, equipped with a halogen lamp, an
Olympus WIB Ðlter set (ex \ 460È490 nm; em \ [515 nm),
and a CCD camera connected to an image recording and pro-
cessing system.
3
2
solved with Et N (0.50 ml, 6.8 mmol) in toluene (10 ml) and
3
the solution was reÑuxed for 1 h. The reaction mixture was
washed with saturated aqueous NaHCO and brine, dried
3
over Na SO , Ðltered, and concentrated. The Ñuorescent oil
2
4
was puriÐed by silica gel column chromatography using
hexaneÈCH Cl to a†ord the desired compound.
2
2
1c. Yield 56%. IR (KBr): l/cm~1 1738 (C2O). 1H NMR (270
Acknowledgements
MHz, CDCl ): d 7.14 (2H, d, J \ 7), 6.98 (2H, d, J \ 7), 5.97
3
This work was supported by Grant-in-Aids for Center of
Excellence (Study of Life Science as Complex Systems) from
the Ministry of Education, Science, Sports and Culture, Japan.
We thank Dr Hiroshi Segawa for helpful discussions on Ñuo-
rescence efficiency measurements.
(2H, s), 4.12 (2H, quar, J \ 7), 4.00 (2H, t, J \ 7), 2.55 (6H, s),
2.29 (2H, t, J \ 7), 1.82 (2H, quin, J \ 7), 1.62 (2H, m), 1.49
(2H, m), 1.44 (6H, s), 1.4È1.2 (10H, m), 1.26 (3H, t, J \ 7 Hz).
3b. Yield 44%. IR (KBr): l/cm~1 1732 (C2O). 1H NMR (270
MHz, CDCl ): d 7.88 (4H, m), 7.46È7.36 (6H, m), 6.70 (2H, s),
3
6.66 (2H, d, J \ 4), 6.55 (2H, d, J \ 4), 4.13 (2H, quar, J \ 7),
4.01 (2H, t, J \ 7), 2.30 (2H, t, J \ 7), 2.21 (6H, s), 1.85 (2H,
quin, J \ 7), 1.63 (2H, m), 1.51 (2H, m), 1.4È1.2 (10H, m), 1.26
(3H, t, J \ 7 Hz).
References
1
2
3
H. Ringsdorf, B. Schlarb and J. Venzmer, Angew. Chem., Int. Ed.
Engl., 1988, 27, 113.
5b. Yield 59%. IR (KBr): l/cm~1 1735 (C2O). 1H NMR (270
MHz, CDCl ): d 7.89 (4H, d, J \ 9), 6.95 (4H, d, J \ 9), 6.69
S. Inoue and K. R. Spring, V ideo Microscopy: the Fundamentals,
2nd edn., Plenum Press, New York, 1997.
3
(2H, s), 6.60 (2H, d, J \ 4), 6.53 (2H, d, J \ 4), 4.12 (2H, quar,
J \ 7), 4.00 (2H, t, J \ 7), 3.83 (6H, s), 2.30 (2H, t, J \ 7), 2.20
(6H, s), 1.85 (2H, quin, J \ 7), 1.63 (2H, m), 1.51 (2H, m), 1.4È
1.2 (10H, m), 1.26 (3H, t, J \ 7 Hz).
R. P. Haugland, Handbook of Fluorescent Probes and Research
Chemicals, 7th edn., Molecular Probes, Eugene, OR, 1999.
A. Treibs and F.-H. Kreuzer, L iebigs Ann. Chem., 1968, 718, 208.
A. Burghart, H. Kim, M. B. Welch, L. H. Thoresen, J. Rei-
benspies, K. Burgess, F. Bergstrom and L. B.-A. Johansson, J.
Org. Chem., 1999, 64, 7813.
F. Li, S. I. Yang, Y. Ciringh, J. Seth, C. H. Martin, D. L. Singh,
D. Kim, R. R. Birge, D. F. Bocian, D. Holten and J. S. Lindsey, J.
Am. Chem. Soc., 1998, 120, 10001.
K. Auwers and E. Borche, Ber. Dtsch. Chem. Ges., 1915, 48, 1698.
S. Bhattacharya, S. De and M. Subramanian, J. Org. Chem., 1998,
63, 7640.
L. H. Thoresen, H. Kim, M. B. Welch, A. Burghart and K.
Burgess, Synlett, 1998, 1276.
4
5
General procedure for the synthesis of 2, 4 and 6. The esters
1, 3 or 5 (0.34 mmol) and KOH (0.80 g, 14 mmol) were dis-
solved in EtOH (10 ml) and the solution was reÑuxed for 1 h.
After cooling, the mixture was neutralized with dilute HCl,
extracted with CHCl and dried over Na SO . The fatty acid
6
7
8
3
2
4
probes were used without further puriÐcation.
2c. Yield 100%. IR (KBr): l/cm~1 1708 (C2O). 1H NMR (270
MHz, THF-d ÈD SO 19: 1): d 7.17 (2H, d, J \ 7), 7.06 (2H,
9
8
2
4
d, J \ 7), 6.00 (2H, s), 4.02 (2H, t, J \ 7), 2.52 (6H, s), 2.21 (2H,
t, J \ 7 Hz), 1.9È1.3 (22H, m).
10 R. L. Hinman and S. Theodoropulos, J. Org. Chem., 1963, 28,
3052.
11 H. Langhals, J. Karolin and L. B.-A. Johansson, J. Chem. Soc.,
4b. Yield 92%. IR (KBr): l/cm~1 1708 (C2O). 1H NMR (270
Faraday T rans., 1998, 94, 2919.
MHz, pyridine-d ): d 8.14 (4H, m), 7.53È7.39 (6H, m), 7.03
5
12 R. D. Kaiser and E. London, Biochim. Biophys. Acta, 1998, 1375,
13.
(2H, d, J \ 4), 6.98 (2H, s), 6.70 (2H, d, J \ 4), 4.09 (2H, t,
J \ 7), 2.53 (2H, t, J \ 7 Hz), 2.28 (6H, s), 1.9È1.7 (4H, m),
1.5È1.2 (12H, m).
13 J. Chen, A. Burghart, A. Derecskei-Kovacs and K. Burgess, J.
Org. Chem., 2000, 65, 2900.
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