Convenient one-pot procedure for synthesizing 4,4′-dimethoxy- boradiaza-s-indacene dyes and their application to cell labeling

Article abstract of DOI:10.1016/j.tet.2011.08.043

We succeeded in developing a convenient one-pot pathway for synthesizing 4,4′-dimethoxy-boradiaza-s-indacene dyes 4a-d. The structures feature two methoxy groups in place of the fluorine atoms in 4,4′-difluoro-4- boradiaza-s-indacene. These novel dyes emi

Full text of DOI:10.1016/j.tet.2011.08.043

Tetrahedron 67 (2011) 7919e7922
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Convenient one-pot procedure for synthesizing 4,40-dimethoxy-boradiaza-s-
indacene dyes and their application to cell labeling
,
,
b
,
c
Kuan-Miao Liu a b, Ming-Shan Tsai a, Ming-Shiou Jan c, Chi-Min Chau a , Wan-Ju Wang
*
a School of Applied Chemistry, Chung Shan Medical University, Taichung 40201, Taiwan, ROC
b Department of Medical Research, Chung Shan Medical University Hospital, Taichung 40201, Taiwan, ROC
c Institute of Microbiology and Immunology, Chung Shan Medical University, Taichung 40201, Taiwan, ROC
a r t i c l e i n f o
a b s t r a c t
We succeeded in developing a convenient one-pot pathway for synthesizing 4,40-dimethoxy-boradiaza-
s-indacene dyes 4aed. The structures feature two methoxy groups in place of the fluorine atoms in 4,40-
difluoro-4-boradiaza-s-indacene. These novel dyes emitted green fluorescence and possessed moderate
Article history:
Received 14 April 2011
Received in revised form 2 August 2011
Accepted 16 August 2011
Available online 23 August 2011
to high fluorescence quantum yields (
F
¼0.32e0.93). We demonstrated that these dyes have applicability
to cell labeling.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
4,40-Dimethoxy-boradiaza-s-indacenes
Trimethyl borate
BODIPY
One-pot
1. Introduction
from the parent, F-BODIPY, and different nucleophiles via nucleo-
philic substitution reactions. In other words, at least two steps are
required to synthesize these BODIPY molecules. Herein, we propose
an alternative one-pot method for synthesizing the O-BODIPY
framework. In our synthesis, the target O-BODIPY molecules were
prepared directly from pyrrole, aldehyde, and trimethyl borate in
one flask. This straightforward approach allowed us to shorten the
Derivatives of 4,40-difluoro-4-boradiaza-s-indacene (F-BODIPY)
have been proven to be useful fluorophores on account of their high
molarabsorptivity, fluorescence quantumyield, and stabilityagainst
light and chemical reactions. Because of these excellent character-
istics, they have also been extensively utilized as biomolecular
labels;1e6 fluorescent chemosensors for metal ions,7e16 protons,17,18
and superoxides;19 and dye-sensitized solar cell (DSSC) material.20
Recently, non-fluorinated BODIPY dyes have been developed rap-
idly and extensively by replacing 4,40-difluorine atoms with other
substituents, such as carbocycle groups (C-BODIPY), ethynyl groups
(E-BODIPY), or alkoxy groups (O-BODIPY).21e23 However, till date,
only a few O-BODIPY dyes have been reported.24,25 Treatment of F-
BODIPY with AlCl3 in dichloromethane and subsequent addition of
alcohol resulted in the total substitution of the two fluorine atoms to
0
ꢀ
afford 4,4 -dialkoxyl-substituted BODIPY, reported by Mely and
Bonnet.26 They found that the O-BODIPY dyes have similar or even
better spectroscopic properties than the parent, F-BODIPY. In this
paper, weprovide an alternativeone-pot procedurefor the synthesis
of 4,40-dimethoxy-boradiaza-s-indacene dyes 4aed, which have
different aryl substituents at the meso position.
As shown in Scheme 1, the BODIPY derivatives C-BODIPY, E-
BODIPY, and O-BODIPY, which are different from each other in
terms of the substituents on the boron atom, are always prepared
Scheme 1. Different types of BODIPY derivatives (C-, E-, and O-BODIPY) and their
* Corresponding author. E-mail address: chimin.chau@gmail.com (C.-M. Chau).
synthetic pathways.
0040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.08.043

7920
K.-M. Liu et al. / Tetrahedron 67 (2011) 7919e7922
reaction procedure and reduce the reaction time by not isolating
the F-BODIPY intermediate, which was a necessary step in the
traditional synthesis of O-BODIPY.
The significantly high quantum yield of compound 4d was at-
tributed to the steric restriction against rotation between the ortho-
methyl phenyl substituent at C8 and the methyl groups at C2 and
C6 of the BODIPY framework. It is well known that steric restriction
increases the dihedral angle between the core and the ortho-methyl
phenyl group and diminishes the excited state energy lost through
nonradiative modes, resulting in enhanced quantum yield and
blue-shifted absorption and emission.27
Because of its high quantum yield, compound 4d is thought to
have potential applications in bioscience. It has the highest quan-
tum yield among these four compounds; therefore, it was selected
to test its potential to bind cells. U937 cells treated with compound
4d were detected by flow cytometry on the basis of green fluo-
rescence on their cell membrane, as shown in Fig.1. DC2.4 and THP-
1 cell lines were also tested, and they showed similar behavior
(Supplementary data).
2. Results and discussion
The general reaction procedure for the synthesis of 4,40-dime-
thoxy-boradiaza-s-indacene by the one-pot pathway is as follows:
2,4-dimethylpyrrole is reacted with aldehyde under trifluoroacetic
acid (TFA) catalysis, which is followed by oxidation of the resulting
dipyrromethane with tetrachloro-p-benzoquinone and, finally, ad-
dition of triethylamine and trimethyl borate. In this experiment, we
used THF as the reaction solvent because tetrachloro-p-benzoqui-
none has low solubility in CH2Cl2. We synthesized the well-known
compound, 4,40-difluoro-8-(4-bromophenyl)-1,3,5,7-tetramethyl-
4-bora-3a,4a-diaza-s-indacene, in dry THF instead of CH2Cl2, which
is the most commonly used solvent in the synthesis of BODIPY, and
found that the yield was much better (33% vs 18%). Therefore, we
selected THF as the solvent throughout the experiment. In addition,
the reaction time, temperature, and quantity of the reagents are
critical to the success of the reaction. A better yield was obtained
when the reaction intermediate, dipyrromethene, was first treated
with Et3N (5 equiv). Green fluorescence was immediately observed;
this was followed by the addition of B(OMe)3 (10 equiv) and
refluxing for 24 h. Another serious problem encountered in the
experiment was that the products were easily hydrolyzed during
silica gel column chromatography. This problem can be solved by
using neutral Al2O3 instead of silica gel and eluting with ethyl ace-
tate/hexane.
Compounds 4aed were obtained in moderate yield and all of
them emitted a green fluorescence (512e516 nm) under excitation
at 500 nm (Scheme 2). The Stokes shift of these four compounds
was not large (11e13 nm), but their fluorescence quantum yields
were moderate to high (0.32e0.93). Table 1 lists the spectroscopic
data of dyes 4aed.
Fig. 1. Compound 4d could bind on cell surface. U937 cells were stained with com-
pound 4d and then determined the green fluorescent intensities using a flow cy-
tometer with 488 nm excitation.
R
In addition, the intracellular binding efficacy was determined by
fluorescent microscopy. As Fig. 2A showing, 4d could label on the
cell surface and intracellular constituents with a dose dependent
manner. Interestingly, 4d was especially bound on cytosolic area
but not nuclei. The mean fluorescent intensity levels of different
staining conditions including group of cell surface labeling with
O
i, ii, iii
R
H
one-pot
N
N
B
O
O
4a : R = 4-bromophenyl (12 %)
4b : R = 4-chlorophenyl (11 %)
4c : R = phenyl (10 %)
4
2
mM 4d (Fig. 2B left panel), or both extra- and intracellular staining
4d : R = 2-methylphenyl (8 %)
with 2 M or 4 M 4d groups (medium and right panels of Fig. 2B)
m
m
were 36.18, 48.03, and 87.52, respectively. These data revealed that
the fluorescent compound 4d had the potential for cell labeling
applications. In the future, we intend to modify the structure of this
dye and to label the desired target specifically.
i
iii
R
R
ii
NH HN
NH
N
3. Conclusions
2
3
In conclusion, we developed a convenient one-pot procedure for
synthesizing new 4,40-dimethoxy-boradiaza-s-indacene dyes
4aed. Among these new dyes, 4d exhibited a very high fluores-
cence quantum yield. The proposed approach is an efficient,
straightforward, and versatile way of synthesizing a new class of
fluorescent BODIPY dyes with potential applications in bio-
chemistry and molecular biology.
Scheme 2. Synthetic pathway and plausible intermediates in the synthesis of O-
BODIPY dyes 4aed. (i) 2,4-dimethylpyrrole, TFA, dry THF; (ii) tetrachloro-p-benzo-
quinone; (iii) Et3N, trimethyl borate, reflux.
Table 1
Absorption and fluorescence data for 4aed in THF
a
Dye
lmax/nm
lem/nmb
FF
Stokes shift/nm
3 )
(Mꢀ1 cmꢀ1
4a
4b
4c
4d
504
504
500
500
515
516
513
512
0.47
0.32
0.48
0.93
11
12
13
12
38,350
37,440
45,870
18,300
4. Experimental
4.1. General materials and methods
a
3-(2-Benzothiazolyl)-7-(diethylamino) coumarin (coumarin-6) in THF
Unless otherwise specified, all reactions were performed under
nitrogen atmosphere using standard Schlenk techniques. THF was
(
FF¼0.99) as the reference.
b
Excited at 500 nm.

K.-M. Liu et al. / Tetrahedron 67 (2011) 7919e7922
7921
Fig. 2. (A) Qualification and (B) quantification of fluorescence of A549 cells after 4d labeling.
distilled from sodium and benzophenone under nitrogen atmo-
sphere. 1H and 13C NMR spectra were recorded on a Varian
400 MHz spectrometer. Absorption and fluorescence spectra were
recorded on a Cary 50 probe UVevis spectrophotometer. All chro-
matographic separations were carried out on MN-Aluminum oxide
(activated neutral, 70e230 mesh). Mass spectra (FAB) were recor-
ded on a VG70-250S mass spectrometer.
(s, 6H), 1.40 (s, 6H); 13C NMR (100 MHz, CDCl3)
d
156.1, 141.0, 139.8,
134.6, 132.6, 132.2, 130.1, 123.0, 121.2, 49.1, 14.8, 14.7; MS (EI) m/z (%)
428.1 (9), 426.1 (9, Mþ), 398.1 (17), 396.1 (100), 381.1 (62), 300.2
(12), 143.1 (23); HRMS (EI) m/z calcd for C21H24BBrN2O2 426.1114,
found 426.1123.
4.2.2. Dimethyl 2-[(Z)-(3,5-dimethyl-2H-pyrrol-2-ylidene)(4-
chlorophenyl)methyl]-3,5-dimethyl-1H-pyrrol-1-yl boronate (4b).
4.2. General procedure for the synthesis of compounds 4aed
Dark brown solid (42 mg, 11%). 1H NMR (400 MHz, CDCl3)
d
7.46 (d,
J¼8.4 Hz, 2H), 7.23 (d, J¼8.4 Hz, 2H), 5.95 (s, 2H), 2.92 (s, 6H), 2.50
(s, 6H), 1.40 (s, 6H); 13C NMR (100 MHz, CDCl3)
156.1, 141.1, 140.0,
2,4-Dimethylpyrrole (2.25 mmol) and aldehyde (1 mmol) [4-
bromobenzaldehyde (1a), 4-chlorobenzaldehyde (1b), benzalde-
hyde (1c), o-tolualdehyde (1d)] were dissolved in dry THF (15 mL)
under nitrogen. One drop of trifluoroacetic acid (TFA) was added and
the solution was stirred at room temperature for overnight
(14e18 h). Tetrachloro-p-benzoquinone (1 mmol) was added once
directly and the mixture was stirred for additional 3 h. The reaction
mixture was then treated with triethylamine (5 mmol) and stirred
for additional 1 h. Trimethyl borate (10 mmol) was added and the
mixture was refluxed for 24 h. After the solutionwas cooled down to
room temperature, H2O (5 mL) was added and the mixture was
extracted with ethyl acetate (80 mL); the organic phase was washed
with water (3ꢁ60 mL) and brine (100 mL), dried over Na2SO4, and
concentrated at reduced pressure. The crudeproductwas purified by
aluminum oxide column chromatography (ethyl acetate/hexane).
d
135.0, 134.1, 132.7, 129.8, 129.3, 121.2, 49.1, 14.7, 14.6; MS (EI) m/z (%)
384.2 (4), 382.2 (12, Mþ), 353.2 (32), 350.2 (100), 335.1 (59), 295.1
(22), 143.1 (10); HRMS (EI) m/z calcd for C21H24BClN2O2 382.1619,
found 382.1614.
4.2.3. Dimethyl 2-[(Z)-(3,5-dimethyl-2H-pyrrol-2-ylidene)(phenyl)
methyl]-3,5-dimethyl-1H-pyrrol-1-yl boronate (4c). Dark brown
solid (35 mg, 10%). 1H NMR (400 MHz, CDCl3)
d
7.46e7.26 (m, 5H),
5.94 (s, 2H), 2.94 (s, 6H), 2.51 (s, 6H), 1.36 (s, 6H); 13C NMR
(100 MHz, CDCl3)
d 155.6, 141.6, 141.2, 135.6, 132.9, 128.9, 128.7,
128.2, 120.9, 49.1, 14.5, 14.4; MS (EI) m/z (%) 348.2 (14, Mþ), 317.2
(81), 316.2 (100), 301.2 (55), 158.6 (12), 143.1 (6); HRMS (EI) m/z
calcd for C21H25BN2O2 348.2009, found 348.2002.
4.2.4. Dimethyl 2-[(Z)-(3,5-dimethyl-2H-pyrrol-2-ylidene)(2-
methylphenyl)methyl]-3,5-dimethyl-1H-pyrrol-1-yl boronate (4d).
Dark brown solid (29 mg, 8%). 1H NMR (400 MHz, CDCl3)
4.2.1. Dimethyl 2-[(Z)-(3,5-dimethyl-2H-pyrrol-2-ylidene)(4-
bromophenyl)methyl]-3,5-dimethyl-1H-pyrrol-1-yl boronate (4a).
Dark brown solid (51 mg, 12%). 1H NMR (400 MHz, CDCl3)
d
7.62 (d,
d
7.34e7.15 (m, 4H), 5.94 (s, 2H), 2.97 (s, 3H), 2.90 (s, 3H), 2.51 (s,
J¼8.0 Hz, 2H), 7.18 (d, J¼8.0 Hz, 2H), 5.95 (s, 2H), 2.92 (s, 6H), 2.50
6H), 2.15 (s, 3H), 1.34 (s, 6H); 13C NMR (100 MHz, CDCl3)
d
155.6,

7922
K.-M. Liu et al. / Tetrahedron 67 (2011) 7919e7922
141.1, 140.1, 135.3, 135.1, 132.4, 130.6, 128.9, 128.0, 126.7, 120.7, 49.1,
49.0, 29.7, 14.6, 13.8; MS (EI) m/z (%) 362.2 (5, Mþ), 330.2 (78), 315.2
(25), 143.1 (9), 91.1 (100), 57.1 (35); HRMS (EI) m/z calcd for
C22H27BN2O2 362.2166, found 362.2168.
References and notes
1. Khatchadourian, A.; Krumova, K.; Boridy, S.; Ngo, A. T.; Maysinger, D.; Cosa, G.
Biochemistry 2009, 48, 5658e5668.
2. Beatty, K. E.; Tirrell, D. A. Bioorg. Med. Chem. Lett. 2008, 18, 5995e5999.
3. Totani, K.; Ihara, Y.; Tsujimoto, T.; Matsuo, I.; Ito, Y. Biochemistry 2009, 48,
2933e2940.
4. Lee, J. S.; Kang, N. Y.; Kim, Y. K.; Samanta, A.; Feng, S.; Kim, H. K.; Vendrell, M.;
Park, J. H.; Chang, Y. T. J. Am. Chem. Soc. 2009, 131, 10077e10082.
5. Ojida, A.; Sakamoto, T.; Inoue, M. A.; Fujishima, S. H.; Lippens, G.; Hamachi, I. J.
Am. Chem. Soc. 2009, 131, 6543e6548.
4.3. General procedure for cell culture and labeling
Cell culture. The U-937, THP-1, and DC2.4 cell lines were cultured
in complete RPMI medium (RPMI-1640 medium; Invitrogen) con-
taining 10% heat-inactivated fetal calf serum (FCS; Biochemical
Industries), 50 U/ml penicillin, and 50 mg/ml streptomycin. The
human lung epithelial cell lines A549 were maintained in Dulbec-
co’s modified Eagle’s medium (DMEM; Invitrogen) supplemented
with 10% FCS, 50 U/ml penicillin, and 50 mg/ml streptomycin. All
the cultures were maintained at 37 ꢂC in 5% CO2 at 95% humidity,
and the medium was changed every 3 days.
Cell labeling. For cell surface labeling, U-937, THP-1 or DC2.4 cells
were washedoncewith PBS and stained with compound 4d onice for
30 min. After washing twice, cells were resuspended in 0.5 mL
FACScan medium (RPMI-1640 medium containing 2% FBS and 0.1%
sodium azide), andthen analyzed usingflowcytometer (FACScan; BD
Biosciences). For intracellular labeling, A549 cells were cultured in
wells of chamber slide (Thermo Scientific, NUNC Lab-TekÔ Chamber
SlideÔ System) at 37 ꢂC in 5% CO2 at 95% humidity for one day and
then fixed with 4% formaldehyde in PBS for 30 min. After washing,
the fixed cells were stained with compound 4d at room temperature
for 30 min and were followed by fluorescence microscopy detection.
6. Sun, Z. N.; Wang, H. L.; Liu, F. Q.; Chen, Y.; Tam, P. K. H.; Yang, D. Org. Lett. 2009,
11, 1887e1890.
7. Yamada, K.; Nomura, Y.; Citterio, D.; Iwasawa, N.; Suzuki, K. J. Am. Chem. Soc.
2005, 127, 6956e6957.
8. Coskun, A.; Akkaya, E. U. J. Am. Chem. Soc. 2005, 127, 10464e10465.
9. Qi, X.; Jun, E. J.; Xu, L.; Kim, S. J.; Joong Hong, J. S.; Yoon, Y. J.; Yoon, J. J. Org.
Chem. 2006, 71, 2881e2884.
€
10. Yu, Y. H.; Descalzo, A.; Shen, Z.; Rohr, H.; Liu, Q.; Wang, Y. W.; Spieles, M.; Li, Y.
Z.; Rurack, K.; You, X. Z. Chem.dAsian J. 2006, 1, 176e187.
11. Coskun, A.; Akkaya, E. U. J. Am. Chem. Soc. 2006, 128, 14474e14475.
12. Mei, Y.; Bentley, P. A.; Wang, W. Tetrahedron Lett. 2006, 47, 2447e2449.
13. Kim, H. J.; Kim, J. S. Tetrahedron Lett. 2006, 47, 7051e7055.
14. Peng, X.; Du, J.; Fan, J.; Wang, J.; Wu, Y.; Zhao, J.; Sun, S.; Xu, T. J. Am. Chem. Soc.
2007, 129, 1500e1501.
15. Kennedy, D. P.; Kormos, C. M.; Burdette, S. C. J. Am. Chem. Soc. 2009, 131,
8578e8586.
16. Wang, Y. W.; Yu, M. X.; Yu, Y. H.; Bai, Z. P.; Shen, Z.; Li, F. Y.; You, X. Z. Tetrahedron
Lett. 2009, 50, 6169e6172.
ꢀ
17. Baruah, M.; Qin, W.; Basaric, N.; De Borggraeve, W. M.; Boens, N. J. Org. Chem.
2005, 70, 4152e4157.
18. Qin, W.; Baruah, M.; De Borggraeve, W. M.; Boens, N. J. Photochem. Photobiol., A
2006, 183, 190e197.
19. Gabe, Y.; Urano, Y.; Kikuchi, K.; Kojima, H.; Nagano, T. J. Am. Chem. Soc. 2004,
126, 3357e3367.
20. Erten-Ela, S.; Yilmaz, M. D.; Icli, B.; Dede, Y.; Icli, S.; Akkaya, E. U. Org. Lett. 2008,
10, 3299e3302.
21. Goze, C.; Ulrich, G.; Ziessel, R. Org. Lett. 2006, 8, 4445e4448.
22. Goze, C.; Ulrich, G.; Mallon, L. J.; Allen, B. D.; Harriman, A.; Ziessel, R. J. Am.
Chem. Soc. 2006, 128, 10231e10239.
23. Goze, C.; Ulrich, G.; Ziessel, R. J. Org. Chem. 2006, 72, 313e322.
24. Kim, H.; Burghart, A.; Welch, M. B.; Reibenspies, J.; Burgess, K. Chem. Commun.
1999, 1889e1890.
Acknowledgements
Financial support from the National Science Council of the Re-
public of China (NSC 99-2113-M-040-003-MY2) and Shin Yuan
Foundation are gratefully acknowledged.
25. Gabe, Y.; Ueno, T.; Urano, Y.; Kojima, H.; Nagano, T. Anal. Bioanal. Chem. 2006,
386, 621e626.
ꢀ
26. Tahtaoui, C.; Thomas, C.; Rohmer, F.; Klotz, P.; Duportail, G.; Mely, Y.; Bonnet,
Supplementary data
D.; Hibert, M. J. Org. Chem. 2007, 72, 269e272.
27. Kee, H. L.; Kirmaier, C.; Yu, L.; Thamyongkit, P.; Youngblood, W. J.; Calder, M. E.;
Ramos, L.; Noll, B. C.; Bocian, D. F.; Scheidt, W. R.; Birge, R. R.; Lindsey, J. S.;
Holten, D. J. Phys. Chem. B 2005, 109, 20433e20443.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tet.2011.08.043.