Convenient method to access new 4,4-dialkoxy- and 4,4-diaryloxy-diaza-s- indacene dyes: Synthesis and spectroscopic evaluation

Article abstract of DOI:10.1021/jo061567m

A straightforward method for the synthesis of original 4,4-dialkoxy- or 4,4-diaryloxy-diaza-s-indacenes (BODIPY) derivatives obtained by treatment of BODIPY 1 with various alcohols in the presence of AlCl₃ is described. The novel compounds are

Full text of DOI:10.1021/jo061567m

number of applications.2 Recently, we synthesized various
BODIPY-labeled pirenzepine derivatives to investigate their
binding to GFP-labeled muscarinic M1 receptors by fluorescence
resonance energy transfer (FRET).3 BODIPY dyes are com-
mercially available4 in small quantities suitable for biochemical
experiments, but amounts typically required for synthetic organic
chemistry are prohibitively expensive. Therefore, as part of our
program to develop fluorescent combinatorial libraries to
identify new ligands of orphan GPCRs by FRET,5 we were
interested in the synthesis of novel and readily accessible
BODIPY-like fluorescent dyes.
In this paper, we describe a rapid and flexible method for
the synthesis of novel 4,4-dialkoxy- or 4,4-diaryloxy-diaza-s-
indacenes 2 by reacting BODIPY 1 with Lewis acids. The
spectroscopic properties of the resulting compounds were similar
or even better than those of the parent BODIPY compound,
highlighting their interest as new fluorescent dyes. Even if recent
articles report on the synthesis of new BODIPY dyes,6 to our
knowledge only one structure of type 2 has been previously
described in the literature.7
Convenient Method To Access New 4,4-Dialkoxy-
and 4,4-Diaryloxy-diaza-s-indacene Dyes:
Synthesis and Spectroscopic Evaluation
Chouaib Tahtaoui,†,|, Ce´cile Thomas,†,| Franc¸ois Rohmer,†
Philippe Klotz,†,§ Guy Duportail,‡ Yves Me´ly,*,‡
Dominique Bonnet,*,† and Marcel Hibert†
De´partement de Pharmacochimie de la Communication
Cellulaire, and De´partement de Pharmacologie et
Physicochimie des Interactions Cellulaires et Mole´culaires,
Institut Gilbert Laustriat, UMR 7175-LC1 ULP/CNRS,
Faculte´ de Pharmacie de Strasbourg, 74 Route du Rhin,
67401 Illkirch, France
dominique.bonnet@pharma.u-strasbg.fr;
yVes.mely@pharma.u-strasbg.fr
ReceiVed July 28, 2006
Synthesis. BODIPY dye 1 has previously been described in
the literature.2f,6e,8 Recently, Golovkova et al. have described a
A straightforward method for the synthesis of original 4,4-
dialkoxy- or 4,4-diaryloxy-diaza-s-indacenes (BODIPY)
derivatives obtained by treatment of BODIPY 1 with various
alcohols in the presence of AlCl3 is described. The novel
compounds are characterized by spectroscopic properties
similar to those of the parent BODIPY 1, absorption and
emission spectra with similar band shapes, high molar
absorption coefficients (ꢀλmax ≈ 80 000 M-1 cm-1), and for
most of them high fluorescence quantum yields (Φexp from
0.52 to 0.71). Among all of the new compounds synthesized,
the dye 2h exhibits higher fluorescence quantum yield (0.71)
and lifetime (4.09 ns) than compound 1 and a good chemical
stability toward conditions compatible with biological cell-
based assays.
(1) Karolin, J.; Johansson, L. B.-A.; Strandberg, L.; Ny, T. J. Am. Chem.
Soc. 1994, 116, 7801.
(2) (a) Wagner, R. W.; Lindsey, J. S. Pure Appl. Chem. 1996, 68, 1373.
(b) Metzker, M. L.; Lu, J.; Gibbs, R. A. Science 1996, 271, 1420. (c)
Malinin, V. S.; Haque, Md. E.; Lentz, B. R. Biochemistry 2001, 40, 8292.
(d) Luedtke, N. W.; Carmichael, P.; Tor, Y. J. Am. Chem. Soc. 2003, 125,
12374. (e) Baruah, M.; Qin, W.; Basaric´, N.; De Borggraeve, W. M.; Boens,
N. J. Org. Chem. 2005, 70, 4152. (f) Golovkova, T. A.; Kozlov, D. V.;
Neckers, D. C. J. Org. Chem. 2005, 70, 5545. (g) Qi, X.; Jun, E. J.; Xu, L.;
Kim, S.-J.; Hong, J. S. J.; Yoon, Y. J.; Yoon, J. J. Org. Chem. 2006, 71,
2881. (h) Ziessel, R.; Bonardi, L.; Retailleau, P.; Ulrich, G. J. Org. Chem.
2006, 71, 3093. (i) Rurak, K.; Resch-Genger, U. Chem. Soc. ReV. 2002,
31, 116.
(3) (a) Ilien, B.; Franchet, C.; Bernard, P.; Morisset, S.; Weill, C. O.;
Bourguignon, J.-J.; Hibert, M.; Galzi, J.-L. J. Neurochem. 2003, 85, 768.
(b) Tahtaoui, C.; Parrot, I.; Klotz, P.; Guillier, F.; Galzi, J.-L.; Hibert, M.;
Ilien, B. J. Med. Chem. 2004, 47, 4300. (c) Bonnet, D.; Ilien, B.; Galzi,
J.-L.; Riche´, S.; Antheaune, C.; Hibert, M. Bioconjugate Chem. 2006, 17,
1618.
(4) (a) Haugland, R. P. The Handbook. A Guide to Fluorescent Probes
and Labeling Technologies, 11th ed.; Invitrogen: Oregon, 2006. (b)
BODIPY is a registred trademark of Molecular Probes, Inc., Eugene, OR.
(5) Hibert, M.; Franchet, C.; Galzi, J. L.; Pattus, F.; Guillier, F. Patent
Application WO 2006003330 (12/01/2006).
(6) (a) Goze, C.; Ulrich, G.; Ziessel, R. Org. Lett. 2006, 8, 4445. (b)
Goze, C.; Ulrich, G.; Mallon, L. J.; Allen, B. D.; Harriman, A.; Ziessel, R.
J. Am. Chem. Soc. 2006, 128, 10231. (c) Ka´lai, T.; Hideg, K. Tetrahedron
2006, 62, 10352. (d) Ulrich, G.; Goze, C.; Guardigli, M.; Roda, A.; Ziessel,
R. Angew. Chem., Int. Ed. 2005, 44, 3694. (e) Chen, J.; Burghart, A.;
Derecskei-Kovacs, A.; Burgess, K. J. Org. Chem. 2000, 65, 2900.
(7) Kim, H.; Burghart, A.; Welch, M. B.; Reibenspies, J.; Burgess, K.
Chem. Commun. 1999, 18, 1889.
4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) dyes
are highly fluorescent molecules1 that have been used for a large
* To whom correspondence should be addressed. Phone: +33(0)390244236.
Fax: +33(0)390244310.
† De´partement de Pharmacochimie de la Communication Cellulaire.
‡ De´partement de Pharmacologie et Physicochimie des Interactions Cellulaires
et Mole´culaires.
§ In fond memory of Dr. Philippe Klotz, our dear friend, colleague, and mentor
who died untimely on June 9th, 2005, aged 40.
| These authors contributed equally to this work.
Present address: Arpida AG, Research & Development of Anti-Infectives,
Dammstrasse 36, CH-4142 Muenchenstein, Switzerland.
10.1021/jo061567m CCC: $37.00 © 2007 American Chemical Society
Published on Web 12/09/2006
J. Org. Chem. 2007, 72, 269-272
269

SCHEME 1a
a Key: (i) IC6H4COCl, CH3MgBr, ether, 81%; (ii) 3, POCl3, CH2Cl2/
pentane, 0 °C, 46%; (iii) IC6H4COOH, POCl3, 24%; (iv) BF3‚OEt2, NEt3,
toluene, 80%.
FIGURE 1. ORTEP view of compound 2a (displacement ellipsoids
at the 50% probability level).
SCHEME 2
two-step process to access compound 1 in a 10% overall yield.2f
Scheme 1 depicts our strategy, which consists of the preparation
of dipyrromethene intermediate 5 and its subsequent reaction
with BF3‚OEt2 in the presence of Et3N in toluene.8b Dipyr-
romethene 5 was obtained following two synthetic routes. In
the first approach, a Grignard reaction of pyrrole 3 in the
presence of 4-iodobenzoyl chloride afforded 2-ketopyrrole 4 in
a good 81% isolated yield.9 Ketopyrrole was subsequently
reacted with pyrrole 3 and phosphorus oxychloride to provide
dipyrromethene 5 in 46% yield.10 The second route investigated
involved the direct condensation of pyrrole 3 with 4-iodobenzene
acid in phosphorus oxychloride used both as solvent and as
reagent. Dipyrromethene 5 was thus obtained in one step but
with a lower yield (24% vs 37%).11 Noteworthy, the isolated
yields were found to be highly dependent from the purity of
the starting pyrroles and POCl3. Prior to the reactions, these
materials were thus freshly distilled.
FIGURE 2. Set of BODIPY dyes 2b-2k obtained by reacting
compound 1 with various alcohols in the presence of AlCl3
(isolated
yield, %).
FIGURE 3. Proposed mechanisms for the formation of BODIPY dyes
2.
Surprisingly, treatment of BODIPY 1 with AlCl3 in dry
dichloromethane and subsequent addition of dry methanol
resulted in a total substitution of the two fluorine atoms to
quantitatively afford molecule 2a (Scheme 2).
Its structure was unambiguously confirmed by single-crystal
X-ray analysis, which showed roughly the same angles between
F-B-N in 1 (110°2′) and O-B-N in 2a (111°55′) (Figure
1).
This compound was found fully stable to air and moisture.
In addition, its spectroscopic properties showed good quantum
yield and molar absorption coefficient (φexp ) 0.52; ꢀλmax
)
79 650 M-1 cm-1) as compared to those of parent BODIPY 1.
This unexpected result prompted us to investigate further the
scope and limitations of the reaction by changing the nature of
both the Lewis acid and the alcohols. Noteworthy, the substitu-
tion of fluorine atoms did not occur in the absence of AlCl3.
Replacing AlCl3 by GaCl3 still allowed one to access compound
2a, whereas reactions with Sc(OTf)3 or TiCl4 led to the
decomposition of BODIPY 1. To establish the scope of our
(8) (a) Chen, J.; Burghart, A.; Wan, C.-W.; Thai, L.; Ortiz, C.;
Reibenspies, J.; Burgess, K. Tetrahedron Lett. 2000, 41, 2303. (b) Burghart,
A.; Kim, H.; Welch, M. B.; Thoresen, L. H.; Reibenspies, J.; Burgess, K.
J. Org. Chem. 1999, 64, 7813.
(9) Wallace, D. M.; Leung, S. H.; Senge, M. O.; Smith, K. M. J. Org.
Chem. 1993, 58, 7245.
(10) Van Koeveringe, J. A.; Lugtenburg, J. Recl. TraV. Chim. Pays-Bas
1977, 96, 55.
(11) Bru¨ckner, C.; Karunaratne, V.; Rettig, S. J.; Dolphin, D. Can. J.
Chem. 1996, 74, 2182.
270 J. Org. Chem., Vol. 72, No. 1, 2007

TABLE 1. Molar Absorption Coefficients (Eλmax) at the Absorption Maximum Wavelengths (λmax), fwhm of Absorption and Emission Spectra,
and Stokes Shift Determined between the Maxima of These Spectra for Compounds 1 and 2a-l in Chloroform Solution at 20 °C
fwhm (cm-1
)
ꢀλmax
M-1 cm-1
λmax
nm
Stokes shift
cm-1
compound
substituent
absorption
emission
1
F
OMe
OEt
OiPr
OBn
OPh
OPh-4-OMe
OPh-4-Cl
OPh-4-NO2
bisnaphthol
catechol
ethyleneglycol
OH
82 150
79 650
79 950
83 800
75 550
80 100
83 300
85 200
79 200
71 050
78 000
52 600
66 950
504
505
505
504
506
506
507
506
505
506
510
507
504
766
766
749
769
803
745
759
736
718
779
764
782
783
855
904
923
900
1004
903
948
887
863
923
526
482
523
526
603
439
437
521
441
398
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
959
950
519
693
TABLE 2. Experimental Fluorescence Quantum Yields (Oexp) and Lifetimes (τ) for Compounds 1 and 2a-2la
τ
τ0
kf
knr
R0
Å
compound
φexp
ns
ns
×108
×108
1
0.57 ( 0.02
0.52 ( 0.02
0.545 ( 0.02
0.67 ( 0.02
0.65 ( 0.02
0.06 ( 0.015
0.004
0.065 ( 0.015
0.71 ( 0.02
0.002
3.11 ( 0.02
2.85 ( 0.05
3.13 ( 0.04
3.79 ( 0.04
3.94 ( 0.03
0.38 ( 0.02
nd
0.37 ( 0.02
4.09 ( 0.02
nd
5.25
5.68
5.45
5.13
5.56
5.73
5.26
5.24
5.67
5.86
nd
1.83
1.82
1.74
1.77
1.65
1.58
nd
1.76
1.74
nd
1.38
1.69
1.45
0.87
0.89
24.74
nd
25.27
0.71
nd
46.0
44.3
44.9
47.6
45.8
31.2
nd
31.7
47.2
nd
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
0
nd
nd
1.51
1.68
nd
9.24
1.44
nd
33.0
41.9
0.14 ( 0.015
0.54 ( 0.02
0.93 ( 0.02
3.21 ( 0.05
7.90
6.14
a The natural radiative fluorescence lifetimes (τ0) were calculated from the Strickler-Berg equation. The radiative kf and nonradiative knr rate constants
were calculated from the experimental quantum yield and lifetime by kf ) φexp/τ and kf + knr ) τ-1, respectively. The Fo¨rster critical distance for homotransfer
(R0) was calculated from the absorption and emission spectra. Experimental conditions are as in Table 1.
reaction, we were next interested in determining the extent to
which the fluorine atoms of BODIPY could be replaced by
various alkyl and aryl alcohols substituted with electron-
withdrawing or -donating groups. Compounds 2a-k were thus
obtained in low to excellent yields (Figure 2). Nevertheless,
the substitution with 1,3-propanediol did not permit one to
access the corresponding cyclic compound.
To shed light on the mechanism of formation of compound
2, some additional experiments were conducted. We hypoth-
esized that AlCl3 activates B-F bonds, thus allowing subsequent
nucleophilic substitution with alcohols (Figure 3).
To reinforce this hypothesis, compound 1 was reacted with
AlCl3 without any alcohol. By TLC monitoring, we observed
the disappearance of the starting BODIPY 1 and the formation
of a more polar product, which was converted rapidly into the
expected compound 2a upon addition of methanol. Attempts
to isolate the polar intermediate by column chromatography on
a deactivated basic alumina failed. The only compound obtained
was the dihydroxy derivative 2l probably formed during the
purification step, because of the presence of water used to
deactivate basic alumina.
NMR studies were also investigated to support this mecha-
nism. 11B NMR analysis (64 MHz) of BODIPY 1 displayed a
triplet pattern due to B-F coupling (δ -2.45 ppm, J ) 32.8
Hz), which evolved upon AlCl3 addition to a broad signal at
5.77 ppm. 19F NMR analysis (188 MHz) confirmed this
observation with a conversion of the quartet observed for
BODIPY 1 (δ -146.75 ppm) to a broad signal at -142.42 ppm.
These results combined with the above studies tend to indicate
that AlCl3 interacts with fluorine atoms to facilitate the
substitution by alcohols.
Spectroscopic Properties. The spectroscopic properties of
all new dyes are collected in Table 1. The introduction of various
alcohols produced only minor changes in the absorption maxima
(λabs ) 504-510 nm). High molar absorption coefficients (ꢀ)
were measured, with values standing around 80 000 M-1 cm-1
,
with only a few exceptions: a higher value was found for
B(OPh-Cl) 2g and lower values for B(bisnaphthol) 2i and
mainly for B(ethyleneglycol) 2k. Absorption and fluorescence
spectra of all compounds showed good mirror symmetry with
similar band shapes for absorption and emission spectra as can
be checked by measurement of their full width at half-maximum
(fwhm). The Stokes shifts were ranging between 400 and 700
cm-1 (11-17 nm). Fluorescence parameters are summarized
in Table 2.
Fluorescence quantum yields (φexp) as well as fluorescence
lifetimes (τ) were first determined experimentally. The fluo-
rescence decays were monoexponential for all compounds,
except for the three of low quantum yield that present a small
additional component (<5%; not presented in Table 2). Radia-
tive lifetimes (τ0) calculated from the Strickler-Berg equation
are in excellent agreement with the inverse of the experimentally
determined radiative rate constant, indicating that neither
interaction of these molecules with the solvent nor change in
J. Org. Chem, Vol. 72, No. 1, 2007 271

their excited-state geometries are occurring. Moreover, while
kf remains relatively constant for all compounds, large differ-
ences appear in their knr values, which thus govern the values
of the fluorescence lifetime and quantum yield. Finally, Fo¨rster
radii for homotransfer were all ranging from 42 and 48 Å, except
for the low quantum yield probes (2e, 2g, and 2k), for which
they are more than 10 Å lower.
In summary, the spectroscopic measurements show that, in
addition to being original constructs, the novel BODIPYs herein
described display interesting fluorescence properties. In par-
ticular, compound 2h exhibits a higher fluorescence quantum
yield (φexp ) 0.71) and lifetime (τ ) 4.09 ns) than those for
BODIPY 1 (0.57 and 3.11 ns). Thus, it represents an interesting
tool with potential application for biological labeling provided
that it displays a good chemical stability toward conditions
compatible with biological cell-based assays. Therefore, BO-
DIPY 2h was dissolved in a Tris-Hcl 50 mM, pH 7.4/MgCl2 5
mM/BSA 1 mg/mL/DMSO 4% mixture at 37 °C. One UV-
visible spectrum per hour was then recorded over a 12 h period,
confirming the excellent stability of compound 2h in this
experimental condition.
In conclusion, we have developed an efficient, rapid, and low-
cost method to access new 4,4-dialkoxy- and 4,4-diaryloxy-
diaza-s-indacene (BODIPY) dyes 2a-l following treatment of
known BODIPY 1 with various alcohols in the presence of
AlCl3. Spectroscopic analysis showed only minor changes in
the absorption maxima and the molar absorption coefficients
as compared to BODIPY 1 and very similar band shapes for
absorption and emission spectra. Among the new structures
synthesized, BODIPY 2h exhibits higher fluorescence quantum
yield and lifetime than BODIPY 1 combined with an excellent
stability in water. Our method represents an efficient, straight-
forward, and versatile entry to a new class of fluorescent
BODIPY dyes with potential applications in biochemistry and
molecular biology.
Experimental Section
General Procedure for the Synthesis of Compounds 2a-l.
BODIPY 1 (200 mg, 0.445 mmol) was dissolved in dry CH2Cl2
(10 mL) in the presence of aluminum chloride (89 mg, 0.668 mmol)
under argon. The resulting mixture was refluxed for 5 min prior to
addition of alcohol (5 mL). After 5 min at room temperature, the
crude mixture was concentrated under reduced pressure, and
compounds 2a-l were isolated by column chromatography on
deactivated basic alumina (CH2Cl2/MeOH 95/5) to afford a red
solid. Data for compound 2a (210 mg, quantitative): 1H NMR
(CDCl3, 300 MHz) δ 7.83 (d, J ) 8.3 Hz, 2H), 7.07 (d, J ) 8.3
Hz, 2H), 5.97 (s, 2H), 2.93 (s, 6H), 2.52 (s, 6H), 1.42 (s, 6H); 13
C
NMR (CDCl3, 50 MHz) δ 156.1, 141.0, 138.2, 135.3, 132.6, 130.3,
121.2, 94.4, 49.1, 14.8, 14.6; 11B NMR (CDCl3, 64 MHz) δ 2.11
(s); ESI-TOF (135 eV) calcd for C21H24BINaN2O2 497.0872, found
497.1193. Anal. Calcd for C21H24BIN2O2: C, 53.20; H, 5.10; N,
5.91. Found: C, 52.80; H, 5.05; N, 5.71.
Acknowledgment. This work was supported by the Centre
National de la Recherche Scientifique, the Universite´ Louis
Pasteur, the Ministe`re de l’Enseignement Supe´rieur et de la
Recherche (C.Th. fellowship), Hoechst-Marion-Roussel (C.Ta.
fellowship), and the European Community’s Sixth Framework
Program (grant LSHB-CT-2003-503337). We are grateful to Dr.
E. Pie´mont for performing fluorescence decay experiments,
Pascale Buisine (IFR85) for ES-MS analyses, and Cyril
Antheaume (IFR85) for NMR experiments.
Supporting Information Available: Crystallographic data for
compound 2a; experimental details and characterizations for
compounds 1, 2a-l, 4, and 5; H and 13C spectra for compounds
1
2a-l; 11B NMR and 19F NMR spectra for compound 1 in the
absence or in the presence of AlCl3; absorption and fluorescence
emission spectra for compounds 1 and 2h; and UV-visible spectra
for the stability study of compound 2h. This material is available
free of charge via the Internet at http://pubs.acs.org.
JO061567M
272 J. Org. Chem., Vol. 72, No. 1, 2007