Long wavelength red fluorescent dyes from 3,5-diiodo-BODIPYs

Article abstract of DOI:10.1039/c001068e

Amphiphilic and long wavelength red fluorescent dyes (4 and 7) were prepared from the Sonogashira coupling reactions of 3,5-diiodo-BODIPYs (1 and 6). One of these compounds, BODIPY 7, readily accumulated within human carcinoma HEp2 cells and was found to

Full text of DOI:10.1039/c001068e

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Long wavelength red fluorescent dyes from 3,5-diiodo-BODIPYs†
Lijuan Jiao,*a Changjiang Yu,a Timsy Uppal,b Mingming Liu,a Yan Li,a Yunyou Zhou,a Erhong Hao,a
Xiaoke Hub and M. Grac¸a H. Vicente*b
Received 19th January 2010, Accepted 6th April 2010
First published as an Advance Article on the web 14th April 2010
DOI: 10.1039/c001068e
Amphiphilic and long wavelength red fluorescent dyes (4 and
7) were prepared from the Sonogashira coupling reactions
of 3,5-diiodo-BODIPYs (1 and 6). One of these compounds,
BODIPY 7, readily accumulated within human carcinoma
HEp2 cells and was found to localize mainly within the
endoplasmic reticulum (ER).
BODIPYs have recently attracted much research interest in diverse
fields,1,2 for example as labeling reagents,1–5 fluorescent switches,6
Fig. 1 Common routes for the functionalizations of the BODIPY
chromophore at the 3,5-positions.
chemosensors,7,8 laser dyes,9 photosensitizers,10 energy transfer
cassettes,11 and harvesting arrays,12 due to their remarkable
properties,2 including large absorption extinction coefficients,
fluorescent BODIPYs. Preliminary in vitro studies on one of these
sharp fluorescence emissions, high fluorescence quantum yields,
high photophysical stability, and low sensitivity to the polarity and
pH of their environment. BODIPYs possessing long wavelength
absorption and emission profiles in the red and near infrared
region (650–900 nm) of the spectrum are particularly promising
for biological applications since the background absorption, light
scattering and the autofluorescence of cell components are largely
reduced.3a
dyes indicate that this type of compound is highly membrane
permeable and it can selectively localize within specific subcellular
organelles, suggesting promising biological applications for these
molecules, namely in bioimaging.
The 3,5-diiodo-BODIPY 1 was obtained in 90% yield upon
BF3·Et2O complexation of 1,9-diiodo-dipyrromethene 2, as
shown in Scheme 1. Dipyrromethene 2 was synthesized from
dipyrromethane 3 using the literature procedure.19 This synthesis
can be scaled up to a multigram scale with minimal chromatogra-
phy isolation. In addition to compound 2, a variety of 1,9-diiodo-
dipyrromethenes are readily available, since dipyrromethanes D
with various pyrrolic functionalities have been widely used in
numerous [2+2] syntheses of porphyrins E.20
Long wavelength absorbing and emitting BODIPYs are of-
ten obtained by extending the conjugation of the BODIPY
chromophore via functionalization of the pyrrolic positions.13
Usually, this is achieved via de novo syntheses from appropri-
ately substituted pyrroles14 (if readily accessible), for example
benzo-/naphtho-fused BODIPYs.15 On another hand, significant
breakthroughs in this area were achieved with the development
of three ready-made BODIPY platforms as shown in Fig. 1: the
3,5-dimethyl-BODIPYs (A),16 the 3,5-dichloro-BODIPYs (B),17
and the 3,5-dithioalkyl-BODIPYs (C).18 These platforms provide
a convenient way for the functionalization of the 3,5-pyrrolic posi-
tions of the chromophore, and have been used to introduce various
functionalities, in particular aryl, alkenyl or alkynyl groups,
therefore conferring desired long wavelength absorption/emission
properties on the BODIPY core. However, BODIPY A can
only react with specific types of aryl adehydes under harsh
conditions, and BODIPYs B and C generally lack b-pyrrolic
substituents. Herein we report an alternative BODIPY platform
complementary to A–C, bearing 3,5-diiodo substituents, and the
use of this platform in Sonogashira coupling reactions leading
to the efficient generation of several long wavelength emitting
Scheme 1 Synthesis of 3,5-diiodoBODIPY 1 from readily available
3. Reaction conditions: (i) Pd/C, H2, THF, 96%; (ii) I2, NaHCO3,
MeOH–H2O, r.t. 80%; (iii) DDQ, 60%; (iv) Et3N, BF3·Et2O, 90%.
The Sonogashira coupling reactions of arylethynes and BOD-
IPY B at 80 ◦C have been reported.17 Consequently, we anticipated
higher reactivity of the 3,5-diiodo-BODIPY platform in this
reaction. BODIPY 1 did show superior reactivity in the Pd(0)-
catalyzed Sonogashira coupling reactions, smoothly generating
the desired long wavelength fluorescent dyes 4a–c as dark blue
solids in 66–67% yields within 2 h at 60 ◦C, as shown in Scheme 2.
Fig. 2 shows normalized absorbance (a) and fluorescence (b)
spectra for compounds 1 and 4a–c. The newly added arylethynyl
aLaboratory of Functional Molecular Solids, Ministry of Education; Anhui
Laboratory of Molecule-Based Materials; School of Chemistry and Ma-
terials Science, Anhui Normal University, Wuhu, Anhui, China, 241000.
E-mail: jiao421@mail.ahnu.edu.cn; Fax: (+86) 553-388-3517
bDepartment of Chemistry, Louisiana State University, Baton Rouge, LA,
70803. E-mail: vicente@lsu.edu; Fax: (+) 225-578-7405
† Electronic supplementary information (ESI) available: The synthesis and
characterization data of all products. See DOI: 10.1039/c001068e
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Scheme
3
Synthesis and functionalization of asymmetric 3,5-di-
iodoBODIPY 6. Reaction conditions: (i) Pd/C, H2, THF, 94%; (ii) I2,
NaHCO3, MeOH–H2O, r.t. 80%; (iii) DDQ, 55%; (iv) Et3N, BF3·Et2O,
80%; (v) 2-ethynylthiophene, Pd(PPh3)2Cl2, CuI, Et3N, THF, 72%.
Scheme 2 Functionalization of 3,5-diiodoBODIPY 1 via the Sonogashira
coupling reaction with arylethynes to generate BODIPYs 4a–c.
groups caused broad absorption bands and red-shifts of both max-
ima, as previously described for 3,5-diarylethynyl-BODIPYs.17
Additional spectroscopic studies were conducted for our com-
pounds, and the results obtained are summarized in Table 1. The
arylethynyl groups caused red-shifts of the maximum absorption
wavelength in the order of 74–90 nm, and maximum fluorescence
wavelength red-shifts in the order of 74–103 nm. The increase
of fluorescent quantum yields was in agreement with previous
reported 3,5-diarylethynyl-BODIPYs.17
and showed similar reactivity in the Sonogashira coupling re-
action as BODIPY 1. Furthermore, BODIPY 6 showed similar
absorption and emission profiles to those of BODIPY 1 (Table 1).
The low fluorescence quantum yields observed for the diiodo-
BODIPYs (1 and 6) can be attributed to the heavy atom effect,
as previously described for 2,6-dibromo-BODIPY16c,23a and 2,6-
diiodo-azaBODIPY,23b both of which have been proposed as
photosensitizers for the photodynamic therapy (PDT)24 of tumors,
due to this effect.
In comparison with the diiodo-BODIPY 6 and as expected, the
newly installed 2-ethynylthiophenes in BODIPY 7 caused large
red-shifts of the absorption and emission maxima (to 116 nm and
110 nm, respectively). Similar results have been observed using
other organic solvents, as shown in Table S1.†
BODIPY 7 was found to rapidly accumulate within human
carcinoma HEp2 cells. Upon incubation with HEp2 cells at 1 mM
concentration for 1 h at 37 ◦C, BODIPY 7 gave bright red
fluorescence, as seen using a Leica DMRXA microscope with
Fig. 2 Normalized UV-vis (a) and fluorescence (b) spectra of BODIPYs
1 (black), 4a (blue), 4b (red) and 4c (green) in dichloromethane.
Interestingly, the newly installed 2-ethynylthiophenes in BOD-
IPY 4a caused significantly larger red-shifts on the maximum
absorption wavelength (90 nm) and maximum fluorescence wave-
length (102 nm) compared with those observed for BODIPY 4b
bearing 3-ethynylthiophene groups (74 nm red-shifts for both
maxima). The nature of the solvent was found to have little effect
on the photophysical properties of these compounds, as shown in
Table S1.†
Under similar optimized reaction conditions as described above,
we also prepared an asymmetric long wavelength BODIPY dye
(7), in 72% yield from the corresponding diiodo-BODIPY, as
shown in Scheme 3. The starting BODIPY 6 was synthesized
in 33% overall yield from readily available dipyrromethane 5,21,22
Table 1 Photophysical properties of BODIPY 1, 4a–c, 6 and 7 in
dichloromethane
BODIPYs
l
max/nm
lge
l
em/nm
Ua
Stokes shift/nm
1
559
649
633
649
558
674
4.66
4.76
4.67
4.77
4.47
4.35
579
681
653
682
576
686
0.07
0.17
0.39
0.29
0.05
0.13
20
12
20
23
18
12
4a
4b
4c
6
Fig. 3 Subcellular localization of BODIPY 7 in HEp2 cells at 1 mM for 1
h: (a) phase contrast, (b) overlay of 7 fluorescence and phase contrast, (c)
LysoSensor Green fluorescence, (e) ER Tracker Blue/White fluorescence,
(g) MitoTracker Green fluorescence, (i) BODIPY FL C5-ceramide fluo-
rescence, and (d, f, h, j) overlays of organelle trackers with 7 fluorescence.
Scale bars: 10 mm.
7
a For BODIPY 1 and 6, the fluorescence quantum yields were calculated
using fluorescein in 0.1 N NaOH aqueous solution (U = 0.90) as the
standard; for BODIPY 4a–c and 7, methylene blue in MeOH (U = 0.03)
was used as the standard.
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40¥ NA 0.8 dip objective lens and DAPI, FITC and Cy5 filter
cubes (see Fig. 3). The overlay with phase contrast (Fig. 3b) shows
that BODIPY has no cytotoxic effect under these conditions. In
order to investigate the subcellular distribution of BODIPY 7, the
HEp2 cells were co-incubated with LysoSensor Green (lysosomes)
at 50 nM for 30 min, ER Tracker Blue/White (ER) at 100 nM for
30 min, MitoTracker Green (mitochondria) at 250 nM for 30 min
and BODIPY FL C5-ceramide (Golgi) at 50 nM for 30 min. The
corresponding overlay images are shown in Fig. 3d, f, h and j. Our
results indicate that the preferential sites of subcellular localization
of BODIPY 7 are the cell ER and also, but to a smaller extent, the
lysosomes. Further biological evaluation of these long wavelength
absorbing/emitting BODIPYs is currently under way.
In summary, long wavelength fluorescent BODIPYs 4 and 7
have been synthesized in good yields from the corresponding 3,5-
diiodo-BODIPYs via Sonogashira coupling reactions. Preliminary
in vitro investigations indicate that these compounds are readily
taken up by cancer cells, have low dark cytotoxicity and accumu-
late preferentially within cell ER. Our results suggest that this type
of BODIPY dye could have promising biological applications.
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Acknowledgements
This work was supported by the National Nature Science Founda-
tion of China (Grants 20802002, 20902004 and 20875002), Anhui
Province (Grants 090416221 and KJ2009A130) and the United
States National Science Foundation (Grant CHE-0911629).
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