Boron-containing two-photon-absorbing chromophores. 3. (1) One-and two-photon photophysical properties of p-carborane-containing fluorescent bioprobes

Article abstract of DOI:10.1021/ic102043v

Boron-containing two-photon-absorbing fluorophores have been prepared as new bifunctional molecules, potentially useful in two-photon excited microscopy (TPEM) and boron neutron capture therapy. They are based on a one-dimensional conjugated system containing a p-carborane entity at one end of the molecule and various electron-donating groups containing oxygen or nitrogen atoms at the other end. We investigated their one- and two-photon photophysical properties. They showed efficient fluorescence in an organic solvent, as well as in water for two of them, allowing microscopy on cell cultures. High two-photon absorption cross sections were determined in the 700-900 nm range. TPEM images were obtained with these new p-carborane-containing fluorophores, with laser intensities in the submilliwatt range.

Full text of DOI:10.1021/ic102043v

ARTICLE
pubs.acs.org/IC
Boron-Containing Two-Photon-Absorbing Chromophores. 3.1
One- and Two-Photon Photophysical Properties
of p-Carborane-Containing Fluorescent Bioprobes
Jean-Franc-ois Nicoud,*,† Frꢀedꢀeric Bolze,† Xiao-Hua Sun,† Ali Hayek,† and Patrice Baldeck‡
†Laboratoire de Biophotonique et Pharmacologie, Universitꢀe de Strasbourg, CNRS (UMR 7213), Facultꢀe de Pharmacie, 74 route du
Rhin, F-67401 Illkirch Graffenstaden Cedex, France
‡Laboratoire de Spectromꢀetrie Physique, Universitꢀe Joseph Fourier, CNRS (UMR 5588), BP 87, F-38402 Saint Martin d’Hꢁeres, France
ABSTRACT: Boron-containing two-photon-absorbing fluoro-
phores have been prepared as new bifunctional molecules,
potentially useful in two-photon excited microscopy (TPEM)
and boron neutron capture therapy. They are based on a one-
dimensional conjugated system containing a p-carborane entity
at one end of the molecule and various electron-donating
groups containing oxygen or nitrogen atoms at the other end.
We investigated their one- and two-photon photophysical
properties. They showed efficient fluorescence in an organic
solvent, as well as in water for two of them, allowing microscopy
on cell cultures. High two-photon absorption cross sections
were determined in the 700ꢀ900 nm range. TPEM images were
obtained with these new p-carborane-containing fluorophores, with laser intensities in the submilliwatt range.
’ INTRODUCTION
new kinds of TPA chromophores in which the interaction
between the two donor moieties proceeds through the cyclodi-
borazane core, which is a pseudoconjugated boron-containing
heterocycle.10 Recently, we have reported the two-photon
photophysical properties of new 1D fluorescent bioprobes
centered on a pyrazabole central core.11 However, the cyclodi-
borazane and pyrazabole heterocycles contain only two boron
atoms, which could be insufficient for efficient BNCT. That is
why we have been considering the introduction of 1,12-dicarba-
closo-dodecaborane (1,12-B10C2H12, known as p-carborane;
Figure 1), which contains 10 boron atoms, to the TPA fluoro-
phores. This unit is known to give derivatives with good thermal
and chemical stability and offers wide possibilities of functiona-
lization. π systems that bear closo-dodecaborate entities have
already been reported by one of us as potential candidates for
TPA processes.12 The introduction of carborane into conjugated
dyads has evidenced the electron-withdrawing characteristics of
carboranes.13 Recently, Tour et al. have prepared new highly
fluorescent BODIPY-based nanocars containing four p-carbor-
ane units, but their TPA properties were not reported.14 So,
p-carborane 1 appears as an interesting building block to investi-
gate novel conjugated fluorescent chromophores containing a
great number of boron atoms and having significant NLO
properties such as TPA.
Nowadays, the conception and synthesis of original nonlinear
optical (NLO) chromophores is a very stimulating area in
organic chemistry, at the interface of chemical engineering,
physics, and, more recently, biology. Indeed, this research field
is based on the multiple potential applications of NLO chromo-
phores, particularly by using multiphoton absorption processes
such as microfabrication,2 3D optical data storage,3 optical
limiting,4 photodynamic therapy,5 or bioimaging.6 For many
years, we have worked on the molecular engineering of efficient
two-photon-absorbing (TPA) fluorophores for uses in in vitro
and in vivo bioimaging.7 Most often the explored fluorophores
consist of a one-dimensional (1D) symmetric conjugated struc-
ture, in which a pair of electron-donor or -acceptor groups are
placed at both extremities, so that an electronic interaction can
occur through the whole 1D molecular conjugated system.8 It
should be noticed, however, that dissymmetric donor/acceptor
systems, well-known in quadratic nonlinear optics, should also be
efficient in cubic nonlinear optics, hence showing significant TPA
properties. In most cases, the electronic interaction proceeds
classically via a π-conjugated system arising from a succession of
aromatic (or heteroaromatic) rings linked by single, double, or
triple bonds. Recently, we have added a new property in such
molecules that could permit one to exploit the two-photon
fluorophores not only in two-photon microscopy but also for
another potential use like therapy. We have chosen to introduce
boron atoms into the structure of TPA fluorophores because
boron-containing drugs are often used as sensitizers in boron
neutron capture therapy (BNCT).9 We have first reported such
These new p-carborane derivatives would lead to bifunctional
molecules, potentially useful in two-photon excited microscopy
Received: October 8, 2010
Published: April 14, 2011
r
2011 American Chemical Society
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|

Inorganic Chemistry
ARTICLE
Chromophore 6a. In a two-necked flask, under an argon atmosphere,
148 mg of 5a (0.39 mmol), 120 mg of 4 (0.19 mmol), 7 mg of PdCl2
(0.04 mmol), 1.19 mg of copper acetate monohydrate (0.006 mmol),
and 41.76 mg of triphenylphosphine (0.16 mmol) were suspended in
50 mL of a THF/triethylamine mixture (15/45, v/v). The mixture was
heated to 80 °C for 12 h, and the solvent was evaporated under vacuum.
The product was purified by column chromatography on alumina, with a
mixture of cyclohexane/dichloromethane (60/40, v/v) as the eluent, to
1
give 6a as a yellow solid (90 mg, 42% yield). H NMR (400 MHz,
Figure 1. Structure of p-carborane 1 (1,12-dicarba-closo-dodecaborane,
B10C2H12).
CDCl3, 25 °C): δ 7.48ꢀ7.37 (m, 6H), 7.33 (d, 2H, J = 8.2 Hz),
7.27ꢀ7.23 (m, 5H), 7.16 (d, 2H, J = 8.6 Hz), 7.07 (d, 4H, J = 8.5 Hz),
7.03 (d, 4H, J = 8.5 Hz), 6.92 (d, 1H, J = 15.9 Hz), 6.32 (d, 1H, J = 15.9
Hz), 5.89 (d, 1H, J = 15.9 Hz), 3.40ꢀ1.21 (m, 11H). 13C NMR (100
MHz, CDCl3, 25 °C): δ 147.65, 147.46, 137.75, 135.01, 132.43, 131.77,
131.72, 131.08, 129.30, 128.34, 126.59, 126.19, 124.62, 123.34, 123.16,
121.62, 90.95, 90.01. 11B NMR (128 MHz, CDCl3, 25 °C): δ 11.7, 13.0,
14.3, 15.6. 11B{H} NMR (128 MHz, CDCl3, 25 °C): δ 12.4, 15.0.
HRMS: m/z616.3913 (calcd for C38H37B10N: m/z 616.3913).
(E)-1,2,3-Tris(dodecyloxy)-5-(4-iodostyryl)benzene (8b). Under an
argon atmosphere, 1 g of 3 (2.80 mmol) was dissolved in anhydrous
THF (5 mL), and then 565 mg of NaH (60% in oil, 14 mmol) was added
slowly. After 1 h of heating at 50 °C, 1.85 g of 3,4,5-tris-
(dodecyloxy)benzaldehyde (7b; 2.80 mmol) in 40 mL of anhydrous
THF was added. The mixture was refluxed for 2 h and quenched with
1 mL of methanol. After cooling, the precipitate was filtered over Celite,
and the solvent was removed under reduced pressure. The crude
product was purified by recrystallization in methanol. The desired
product 8b was obtained as a white solid (2.04 g, 85% yield). 1H
NMR (400 MHz, CDCl3, 25 °C): δ 7.66 (d, 2H, J = 8.3 Hz), 7.23 (d, 2H,
J = 8.3 Hz), 7.03 (d, 1H, J = 16.2 Hz), 6.85 (d, 1H, J = 16.2 Hz), 6.70 (s,
2H), 4.04ꢀ3.95 (m, 6H), 1.87ꢀ1.71 (m, 6H), 1.50ꢀ1.48 (m, 6H)
1.36ꢀ1.27 (m, 48H), 0.89 (t, 9H, J = 6.4 Hz). 13C NMR (100 MHz,
CDCl3, 25 °C): δ 153.29,138.55, 137.66, 136.90, 132.04, 129.69, 128.02,
126.42, 105.26, 92.41, 73.49, 69.15, 31.91, 31.89, 30.30, 29.72, 29.70,
29.68, 29.66, 29.62, 29.58, 29.39, 29.33, 26.08, 22.66, 14.08. HRMS: m/z
858.5368 (calcd for C50H83IO3: m/z 858.5386).
(TPEM) and BNCT. The TPA imaging capabilities and BNCT
sensitizing ambivalence could be of great interest for studying the
mechanism of the processes involved and allowing an accurate
localization of the sensitizer during the therapy.15 Such molecules
(as boron-containing porphyrins) are in growing emphasis.16
TPEM takes advantage of the TPA properties in living tissues and
animals, such as deep penetration or low photodamage.17
Intravital TPEM was used to study the effects of tomographic
synchrotron irradiation on mouse brain microvasculature, point-
ing out the interest of this microbeam radiation therapy.18 We
report here the synthesis and characterization of new 1D donor/
acceptor TPA chromophores 6aꢀ6c containing a p-carborane
moiety at one end and bearing various donor groups at the other
end that could be used as such bifunctional molecules. Pre-
liminary TPEM images will also be presented.
’ EXPERIMENTAL SECTION
Synthesis. All chemicals and reagents were purchased from Aldrich
or Acros Organics and were used as received unless specified. The
starting material p-carborane 1 was obtained from Katchem Inc. Tetra-
hydrofuran (THF) was distilled over sodium under an argon atmo-
sphere prior to use. 1H, 13C, and 11B NMR spectra were recorded with a
Bruker USþ 400 instrument (BBFOþ probe) in CDCl3. The chemical
shifts for the 1H and 13C NMR spectra are reported in ppm at 400.13 and
100.63 MHz. Spectra are referenced internally to residual protic solvent
(E)-1,2,3-Tris[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]-5-(4-iodostyryl)-
benzene (8c). Under an argon atmosphere, 660 mg of 3 (1.86 mmol)
was dissolved in anhydrous THF (20 mL), and then 565 mg of NaH
(60% in oil, 14 mmol) was added slowly. After 1 h of heating at 50 °C,
1.22 g of 3,4,5-tris[2-(2-methoxyethoxy)ethoxy]benzaldehyde (7c; 1.86
mmol) in 20 mL of anhydrous THF was added. The mixture was
refluxed for 2 h and quenched with 1 mL of methanol. After cooling, the
precipitate was filtered over Celite, and the solvent was removed under
reduced pressure. The crude product was purified by column chroma-
tography on silica gel with dichloromethane/methanol ratios of 97/3 to
50/50 (v/v) as the eluent. The desired product 8c was obtained as a
yellow oil (1.10 g, 76% yield). 1H NMR (400 MHz, CDCl3, 25 °C): δ
7.66 (d, 2H, J = 8.3 Hz), 7.22 (d, 2H, J = 8.3 Hz), 7.15ꢀ6.83 (d, 1H, J =
16.2 Hz), 6.83 (d, 1H, J = 16.2 Hz), 6.74 (s, 2H), 4.22ꢀ4.08 (m, 6H),
3.86 (t, 4H, J = 5.5 Hz), 3.79 (t, 2H, J = 5.5 Hz), 3.75ꢀ3.62 (m, 18H),
3.55ꢀ3.52 (m, 6H), 3.36 (s, 9H). 13C NMR (100 MHz, CDCl3, 25 °C):
δ 152.75, 138.68, 137.65, 136.71, 132.39, 129.22, 128.61, 128.04, 126.84,
126.34, 106.41, 92.57, 72.33, 71.88, 71.87, 70.76, 70.64, 70.62, 70.50,
70.48, 70.46, 69.71, 68.87, 67.88, 58.95. HRMS: m/z 810.29248 (calcd
for C35H57INO12: m/z 810.29254, M þ NH4).
(CHCl3, δ 7.26) for 1H NMR spectra and to CDCl3 (δ 77 ppm) for 13
C
NMR spectra. The chemical shifts for 11B NMR spectra are reported in
ppm at 128.37 MHz. Mass spectra were recorded with either a Bruker
MicrOTOF-Q (ESI), an Agilent MSD (MM-ES-ACPI), or a Bruker
Autoflex (MALDI-TOF) spectrometer. Thin-layer chromatography was
run on Merck precoated aluminum plates (Silica 60 F254). Column
chromatography was run on Merck silica gel (60ꢀ120 mesh) or Merck
neutral alumina (70ꢀ230 mesh). Compounds 2,19 3,10 and 5a10 were
prepared as previously described.
(E)-C-4-iodostyryl-p-carborane (4). Under an argon atmosphere,
diethyl (4-iodobenzyl)phosphonate (3; 70 mg, 0.19 mmol) was dis-
solved in 30 mL of anhydrous THF, and then 10 mg of NaH (60% in oil,
0.25 mmol) was added. After the effervescence ceased, the suspension
was refluxed for 1 h. C-monoformyl-p-carborane (2) in 20 mL of
anhydrous THF was added dropwise (25 mg, 0.16 mmol), and then
the mixture was refluxed for 2 h. After filtration, the solvent was
evaporated under vacuum. The crude product was purified by column
chromatography on silica gel with dichloromethane as the eluent, to give
4 as a white solid (40 mg, 70% yield).
(E)-Trimethyl[[4-[3,4,5-tris(dodecyloxy)styryl]phenyl]ethynyl]silane
(9b). In a three-necked flask, 0.08 mL of (trimethylsilyl)acetylene (51.9
mg, 0.53 mmol), 500 mg of 8b (0.63 mmol), 24.51 mg of Pd(PPh3)2Cl2
(0.035 mmol), and 10.2 mg of CuI (0.052 mmol) were suspended in
30 mL of anhydrous THF and 5 mL of isopropylamine. The mixture was
deaerated with argon and then sonicated for 30 min at room tempera-
ture. The solvents were evaporated under vacuum, and the crude
product was purified by column chromatography on alumina with
1H NMR (400 MHz, CDCl3, 25 °C): δ 7.59 (d, 2H, J = 8.4 Hz), 6.98
(d, 2H, J = 8.4 Hz), 6.27 (d, 1H, J = 15.5 Hz), 5.85 (d, 1H, J = 15.5 Hz),
3.10ꢀ1.21 (m, 11H). 13C NMR (100 MHz, CDCl3, 25 °C): δ 137.7,
134.7, 131.0, 128.3, 127.5, 93.9, 58.9. 11B NMR (128 MHz, CDCl3,
25 °C): δ 11.8, 13.1, 14.3, 15.6. 11B{H} NMR (128 MHz, CDCl3,
25 °C): δ 12.4, 15.0. HRMS: m/z 372.13845 (calcd for C10H17B10I: m/z
372.13781).
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ARTICLE
97/3 (v/v) cyclohexane/methanol as the eluent, to yield the desired
product 9b as a white solid (360.1 mg, 82% yield). 1H NMR (400 MHz,
CDCl3, 25 °C): δ 7.45 (d, 2H, J = 8.8 Hz), 7.40 (d, 2H, J = 8.8 Hz), 7.06
(d, 1H, J = 16.2 Hz), 6.90 (d, 1H, J = 16.2 Hz), 6.70 (s, 2H), 4.04ꢀ3.95
(m, 6H), 1.85ꢀ1.73 (m, 6H), 1.54ꢀ1.44 (m, 6H), 1.27 (m, 48 H), 0.88
(t, 9H, J = 6.3 Hz), 0.27 (s, 9H). 13C NMR (100 MHz, CDCl3, 25 °C): δ
153.28, 138.59, 137.88, 132.39, 132.09, 130.08, 126.74, 126.13, 120.73,
105.31, 80.65, 73.50, 69.16, 31.90, 31.88, 30.30, 29.71, 29.69, 29.66,
29.62, 29.57, 29.39, 29.35, 29.32, 26.08, 22.64, 14.07, ꢀ0.05. HRMS: m/
z 829.147 (calcd for C55H92SiO3: m/z 829.403).
(E)-Trimethyl[[4-[3,4,5-tris[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]-
styryl]phenyl]ethynyl]silane (9c). In a three-necked flask, 0.09 mL of
(trimethylsilyl)acetylene (62 mg; 0.63 mmol), 500 mg of 8c (0.63 mmol),
26 mg of Pd(PPh3)2Cl2 (0.037 mmol), and 12 mg of CuI (0.063 mmol)
were suspended in 30 mL of anhydrous THF and 5 mL of isopropyla-
mine. The mixture was deaerated with argon and sonicated for 30 min at
room temperature. The solvents were evaporated under vacuum, and
the crude product was purified by column chromatography on alumina
with 90/10 (v/v) cyclohexane/methanol as the eluent, to yield the
desired product 9c as a colorless oil (408 mg, 85% yield). 1H NMR (400
MHz, CDCl3, 25 °C): δ 7.46 (d, 2H, J = 8.5 Hz), 7.39 (d, 2H, J = 8.5 Hz),
7.02 (d, 1H, J = 16.2 Hz), 6.89 (d, 1H, J = 16.2 Hz), 6.75 (s, 2H),
4.22ꢀ4.15 (m, 6H), 3.87 (t, 4H, J = 5.3 Hz), 3.79 (t, 2H, J = 5.3 Hz),
3.76ꢀ3.61 (m, 18H), 3.56ꢀ3.53 (m, 6H), 3.37 (s, 3H), 3.37 (s, 6H),
0.25 (s, 9H). 13C NMR (100 MHz, CDCl3, 25 °C): δ 152.74, 138.65,
137.32, 132.57, 132.25, 129.38, 128.63, 127.34, 126.36, 126.11, 121.94,
106.44, 105.12, 95.02, 72.35, 71.89, 70.77, 70.66, 70.63, 70.52, 70.49,
70.46, 69.71, 68.86, 58.97, 53.37, ꢀ0.05. HRMS: m/z 762.816 (calcd for
C40H62SiO12: m/z 762.999).
(E)-1,2,3-Tris(dodecyloxy)-5-(4-ethynylstyryl)benzene (5b). Under
an argon atmosphere, 400 mg of 9b (0.55 mmol) was dissolved in
20 mL of THF, and then 0.28 mL of 1 M tetrabutylammonium fluoride
(TBAF) was added. The mixture was stirred at room temperature for
1 h. The solvents were evaporated under vacuum, and the crude product
was purified by column chromatography on silica gel with cyclohexane as
the eluent. The pure product 5b was obtained as a yellow oil (356 mg,
98% yield). 1H NMR (400 MHz, CDCl3, 25 °C): δ 7.49 (d, 2H,
J = 8.77 Hz), 7.43 (d, 2H, J = 8.77 Hz), 7.02 (d, 1H, J = 16.2 Hz), 6.92 (d,
1H, J = 16.2 Hz), 6.71 (s, 2H), 4.04ꢀ3.95 (m, 6H), 3.13 (s, 1H),
1.87ꢀ1.70 (m, 6H), 1.54ꢀ1.44 (m, 6H), 1.27 (m, 48H), 0.88 (t, 9H).
13C NMR (100 MHz, CDCl3, 25 °C): δ 153.28, 138.59, 137.88, 132.39,
132.09, 130.08, 126.74, 126.13, 120.73, 105.31, 83.71, 73.50, 69.16,
31.90, 31.88, 30.30, 29.71, 29.69, 29.66, 29.62, 29.57, 29.39, 29.35, 29.32,
26.08, 22.64, 14.07. HRMS: m/z 756.64205 (calcd for C52H84O3: m/z
756.64265).
(E)-1,2,3-Tris[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]-5-(4-ethynyl-
styryl)benzene (5c). Under an argon atmosphere, 420 mg of 9c (0.55
mmol) was dissolved in 20 mL of THF, and then 0.19 mL of 1 M TBAF
was added. The mixture was stirred at room temperature for 1 h. The
solvent was evaporated under vacuum, and the crude product was
purified by column chromatography on silica gel with cyclohexane as the
eluent. The pure product 5c was obtained as a yellow oil (372 mg, 98%
yield). 1H NMR (400 MHz, CDCl3, 25 °C): δ 7.49 (d, 2H, J = 8.8 Hz),
7.42 (d, 2H, J = 8.8 Hz), 7.02 (d, 1H, J = 16.2 Hz), 6.94 (d, 1H, J = 16.2
Hz), 6.78(s, 2H), 4.23ꢀ4.15 (m, 6H), 3.85 (t, 4H, J = 4.8 Hz), 3.79 (t,
2H, J = 4.8 Hz), 3.76ꢀ3.61 (m, 18H), 3.56ꢀ3.53 (m, 6H), 3.37 (s, 3H),
3.37 (s, 6H), 3.13 (s, 1H). 13C NMR (100 MHz, CDCl3, 25 °C): δ
152.77, 138.74, 137.68, 132.49, 132.41, 129.61, 127.20, 126.36, 126.19,
120.89, 106.52, 83.68, 72.36, 71.89, 70.78, 70.66, 70.64, 70.52, 70.49,
70.47, 69.73, 68.89, 58.98. HRMS: m/z 690.3615 (calcd for C37H54O12:
m/z 690.36257).
and 30 mg of triphenylphosphine (0.11 mmol) were suspended in
60 mL of THF/triethylamine (15/45, v/v). The mixture was heated to
80 °C for 12 h, and the solvent was evaporated under vacuum. The
product was purified by column chromatography on alumina, with a
mixture of cyclohexane/dichloromethane (50/50, v/v) as the eluent, to
give 6b as a pale-yellow solid (100 mg, 37% yield). 1H NMR (400 MHz,
CDCl3, 25 °C): δ 7.47 (d, 2H, J = 8.3 Hz), 7.44 (d, 2H, J = 8.3 Hz), 7.41
(d, 2H, J = 8.3 Hz), 7.2 (d, 2H, J = 7.3 Hz), 7.02 (d, 1H, J = 15.3 Hz), 6.93
(d, 1H, J = 15.3 Hz), 6.69 (s, 2H), 6.32 (d, 1H, J = 15.8 Hz), 5.89 (d, 1H,
J = 15.8 Hz), 4.01 (t, 4H, J = 6.5 Hz), 3.96 (t, 2H, J = 6.5 Hz), 3.40ꢀ2.10
(m, broad, 11H), 1.78 (m, 6H), 1.47 (m, 6H), 1.25 (m, 48H), 0.87 (t,
9H, J = 6.6 Hz). 13C NMR (100 MHz, CDCl3, 25 °C): δ 153.36, 138.68,
137.45, 135.07, 132.36, 132.18, 131.92, 131.80, 129.96, 127.77, 127.50,
126.61, 126.30, 123.30, 121.85, 105.42, 90.89, 90.11, 73.58, 69.25, 31.96,
30.30, 29.74, 29.62, 19.40, 29.33, 26.16, 22.72, 14.13. 11B NMR (128
MHz, CDCl3, 25 °C): δ 11.7, 13.0, 14.3, 15.6. 11B{H} NMR (128 MHz,
CDCl3, 25 °C): δ 12.3, 15.0. HRMS: m/z 1001.8706 (calcd for
C62H100O3B10: m/z 1001.8676).
Chromophore 6c. In a two-necked flask, under an argon atmosphere,
300 mg of 5c (0.43 mmol), 130 mg of 4 (0.21 mmol), 18 mg of
Pd(PPh3)2Cl2 (0.026 mmol), and 8.25 mg of copper iodide (0.0043
mmol) were suspended in 50 mL of THF/triethylamine (15/45, v/v).
The mixture was sonicated for 2 h and stirred for 2 days. The solvents
were then evaporated under vacuum. The product was purified by
column chromatography on alumina, with a mixture of cyclohexane/
methanol (90/10, v/v) as the eluent, to give 6c as a yellow oil (167 mg,
45% yield). 1H NMR (400 MHz, CDCl3, 25 °C): δ 7.47 (d, 2H, J = 9.1
Hz), 7.43 (d, 2H, J = 9.1 Hz), 7.41 (d, 2H, J = 7.4 Hz), 7.19 (d, 2H, J = 8.4
Hz), 6.99 (d, 1H, J = 15.7 Hz), 6.92 (d, 1H, J = 15.7 Hz), 6.74 (s, 2H),
6.32 (d, 1H, J = 15.7 Hz), 5.88 (d, 1H, J = 15.7 Hz), 4.19 (t, 4H, J = 5.4
Hz), 4.15 (t, 2H, J = 4.9 Hz), 3.85 (t, 4H, J = 5.4 Hz), 3.77 (t, 2H, J = 4.5
Hz), 3.71 (m, 6H), 3.64 (m, 12H), 3.52 (m, 6H), 3.35 (s, 3H), 3.34 (s,
6H), 3.2ꢀ2.21 (m, broad, 11H). 13C NMR (100 MHz, CDCl3, 25 °C):
δ 152.7, 138.4, 137.27, 135.08, 132.83, 132.41, 131.93, 131.79, 129.36,
128.70, 128.35, 127.56, 126.55, 126.60, 126.37, 123.25, 122.03, 106.50,
106.38, 90.83, 90.12, 83.03, 72.35, 71.90, 71.85, 70.75, 70.63, 70.51,
70.44, 70.41, 69.68, 68.85, 59.04. 11B NMR (128 MHz, CDCl3, 25 °C):
δ 11.8, 13.0, 14.3, 15.6. 11B{H} NMR (128 MHz, CDCl3, 25 °C): δ
12.4, 15.0. HRMS: m/z 958.5745 (calcd for C47H70O12B10: m/z
958.5761).
One-Photon Photophysics. UVꢀvisible absorption spectra were
recorded on a Cary 400 spectrophotometer in a dual-beam mode using a
matched pair of 1 ꢁ 0.5 cm quartz cells. Pure solvent was used as the
reference. Values of the molar extinction coefficients, ελmax, of the
compounds of interest were obtained as the average of at least five
independent measurements with absorbance in the range 0.5ꢀ1.
Fluorescence emission and excitation spectra were performed on a
Fluorolog (Jobin-Yvon) spectrofluorimeter with optically dilute solu-
tions (Abs. < 0.15) in 1 ꢁ 0.5 cm cells. Fluorescence quantum yields
were measured by the relative method, using recrystallized quinine
sulfate in 0.5 M aqueous H2SO4 (j = 0.54)20 as the reference. For these
measurements, the slit widths were adjusted so that the spectral
bandwidths of the absorption and emission were identical at 1.0 nm,
and the absorbances of the sample and the reference were chosen so that
they were in the 0.1ꢀ0.15 range and nearly identical at the same
excitation wavelength. Emission quantum yields were then calculated
according to the method described by Crosby and Demas, taking into
account the differences between the refractive indices of the sample and
reference solutions.20 Fluorescence lifetime measurements were per-
formed using the time-correlated single-photon-counting technique,
according to the method described by Lami and Piꢀemont.21
Chromophore 6b. In a two-necked flask, under an argon atmosphere,
220 mg of 5b (0.29 mmol), 87 mg of 4 (0.14 mmol), 5 mg of PdCl2
(0.028 mmol), 0.85 mg of copper acetate monohydrate (0.004 mmol),
Two-Photon Excitation Characterizations. The TPA cross-
sectional spectra were obtained by up-conversion fluorescence measure-
ments using a Ti:sapphire femtosecond laser in the range 700ꢀ900 nm.
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The excitation beam is collimated over the cell length (10 mm). The
fluorescence, collected at 90° of the excitation beam, was focused into an
optical fiber connected to a spectrometer. The incident beam intensity
was adjusted to ensure an intensity-squared dependence of the fluores-
cence over the whole spectral range. Calibration of the spectra was
performed by comparison with p-bis(o-methylstyryl)benzene, for which
σ2 = 70 GM at 570 nm, and with the published 700ꢀ900 nm Rhodamine
B TPA spectrum.22
Bioimaging. One- and two-photon excited microscopies were
performed on a Leica TCS SP2 AOBS MP microscope. For one-photon
images, the excitation source was a continuous wave 405 nm laser diode,
and for two-photon images, the excitation was performed by a Spectra
Physics Tsunami laser coupled to the microscope by an optical fiber
(repetition rate of 80 MHz and pulse duration of 1.5 ps). We used a 63ꢁ
oil immersion objective HCX PL APO CS Lambda Blue (NA = 1.4).
’ RESULTS AND DISCUSSION
Synthesis. We have prepared three 1D chromophores 6aꢀ6c
containing at one end an acceptor p-carborane unit and at the
other end various donor groups. All three are well-soluble in
organic solvents, while only 6c is sufficiently water-soluble to
allow photophysical characterizations in water (Figure 2). The
commercially available p-carborane 1 (B10C2H12) was functio-
nalized by monoformylation with 1 equiv of methyl formate to
give the C-formylcarborane 2 in 65% yield. The reaction of 1
equiv of 3 with the monoformyl derivative 2 affords (E)-4-
iodostyryl-p-carborane moiety 4 in 70% yield (Scheme 1). This
compound can then be used as the starting material for the
classical palladium cross-coupling reactions in order to build the
targeted 1D conjugated chromophores 6aꢀ6c.
The iodo derivative 4 was coupled to three different con-
jugated building blocks bearing donor groups (Scheme 2):
(E)-4-(4-ethynylstyryl)-N,N-diphenylaniline (5a) and 5b to give
chromophores 6a and 6b, which are soluble in organic solvents,
and 5c bearing three short oligo(ethylene glycol) (OEG) chains
to give the nonionic water-soluble chromophore 6c. The donor
building block 5a was prepared as described previously.10
The synthetic pathway for the other oxygen-containing donor
groups is shown in Scheme 3. All compounds were fully
Figure 2. Absorption (black) and emission (red) spectra of 6a (a), 6b
(b), and 6c (c) in dichloromethane. Absorption (blue) and emission
(green) spectra of 6c in water (c).
Scheme 1. Synthesis of 4a
a (i) BuLi, methyl formate, and then 5 M HCl. (ii) THF, NaH, 1 h, reflux.
Scheme 2. Synthesis of chromophores 6aꢀ6ca
a (i) PdCl2, Cu(OAc)2(H2O), PPh3, and THF/Et3N for 6a and 6b and Pd(PPh3)2Cl2, CuI, and THF/Et3N for 6c.
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Scheme 3. Preparation of Oxygen-Containing Donor-Conjugated Systems 5b and 5ca
a (i) NaH, THF. (ii) (Trimethylsilyl)acetylene, Pd(PPh3)2Cl2, CuI, THF/Et3N, and ultrasound. (iii) TBAF and THF at room temperature.
Table 1. Photophysical Properties of Chromophores 6aꢀ6c in Dichloromethane and Water
absorption
emission
H2O
CH2Cl2
H2O
λmax (nm)
CH2Cl2
λmax (nm)
chromophore
λmax (nm)
ε ꢁ 10ꢀ4 (Mꢀ1 cmꢀ1
)
λmax (nm)
ε ꢁ 10ꢀ4 (Mꢀ1 cmꢀ1
)
Φ (%)
Φ (%)
6a
6b
6c
395
363
360
9.9
12.2
10.7
496
458
438
58
59
69
346
7.2
467
24
1
characterized by H, 13C, and 11B NMR spectroscopy, UVꢀ
visible spectroscopy, and high-resolution mass spectrometry
(HRMS).
Photophysical Properties. The one-photon photophysical
properties of chromophores 6aꢀ6c were studied in solution in
dichloromethane, and 6c was also studied in water. In an organic
solvent, all three chromophores present an intense absorption
maximum in the 360ꢀ395 nm range. The values of λmax and the
corresponding ε are presented in Table 1.
The UVꢀvisible and emission spectra are depicted in Figure 2.
For chromophores 6b and 6c, those bearing alkoxy and OEG
donor groups, the shapes of the UVꢀvisible and emission spectra
are similar in dichloromethane (229 cmꢀ1 shift in λmax values and
1.5 ꢁ 104 Mꢀ1 cmꢀ1 difference for εmax). For chromophore 6a,
bearing the diphenylamino donor group, we notice a 2461 cmꢀ1
red shift of λmax compared to the oxygenated chromophores 6b
and 6c, as is expected for a better electron-donating group.
Because chromophore 6c is water-soluble, its photophysical
properties were also investigated in water. There is a slight blue
shift of 1124 cmꢀ1 from dichloromethane to water. In addition,
the molar extinction coefficient εmax decreases from 10.7 ꢁ
104 Mꢀ1 cmꢀ1 in dichloromethane to 7.2 ꢁ 104 Mꢀ1 cmꢀ1 in water.
In dichloromethane, chromophores 6b and 6c present a blue
fluorescence, while 6a emits in the green region. Emission data
are presented in Table 1. We can observe a blue shift in the
emission from 6b to 6c, indicating a difference between the two
chromophores in their excited states. In water, the emission of
chromophore 6c is red-shifted by 1418 cmꢀ1 in association with
Figure 3. Two-photon excitation spectra of chromophores 6a (blue),
6b (red), and 6c (green) in dichloromethane.
a decrease of the fluorescence quantum yield compared to those
measured in dichloromethane. Fluorescence lifetimes were also
determined in dichloromethane. The three fluorophores present
a fluorescence monoexponential decay, leading to lifetimes of
1.60, 1.25, and 1.17 ns for 6aꢀ6c respectively. In water, 6c
presents a multiexponential decay, leading to lifetimes of 0.12 ns
(27%), 0.72 ns (41%), 2.32 ns (26%), and 6.35 ns (6%).
The two-photon photophysical properties of these fluoro-
phores were investigated by way of their two-photon excited
fluorescence. The two-photon excitation spectra are depicted
in Figure 3. Chromophore 6a presents a large and strong
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Figure 4. CLSM of HeLa cells dyed with fluorophores 6a (left) and 6c (right).
Figure 5. TPEM images of HeLa cell stained with fluorophores 6a (left) and 6c (right). Excitation wavelength at 800 nm (laser power for 6a, 1ꢀ2 mW,
and for 6c, 15 mW).
two-photon absorption band in the wavelength region studied
(700ꢀ900 nm) with a maximum σ2 of 470 GM at 810 nm, while
for chromophores 6b and 6c, only the absorption tails are
observed. The TPA maxima are located under 700 nm, when
the σ2 values are 300 and 200 GM at 700 nm for 6b and 6c,
respectively (1 GM = 10ꢀ50 cm4 s photonꢀ1).
Bioimaging. Our aim was to check the behavior of 6a and 6c
during preliminary fluorescence microscopy investigations.
Fluorophore 6b could not be used because of its insolubility in
water. Fluorophore 6c was readily soluble in an Hank’s Balanced
Salt Solution (HBSS) buffer (10ꢀ4 M). We have found that
fluorophore 6a was also soluble in HBSS in the presence of 1%
dimethyl sulfoxide (for a final concentration of ∼10ꢀ5 M),
allowing biological studies.
Dyes 6a and 6c were first studied by confocal laser scanning
microscopy (CLSM) onaLeicaLSP2. Theywereinternalizedvery
rapidly (<5 min), but the localization behaviors of these two dyes
were very different. Figure 4 shows their localization in HeLa cells
(superposition of fluorescence and bright-field images). For 6a, the
distribution is heterogeneous, with visible vesicles, and for 6c, the
distribution is much more diffuse because of better water solubility.
TPEM was then tested, with an excitation wavelength of 800 nm.
This wavelength lies in the most efficient domain of commercial laser
microscopes (stability and power). With dye 6c, the signal is low
because of the mismatch in the excitation wavelength, while for 6a,
the signal is very intense down to the 1ꢀ2 mW excitation power
range at the specimen (Figure 5). The distributions are similar to that
obtained by one-photon excitation.
’ CONCLUSIONS
Three new 1D fluorescent boron-containing TPA chromo-
phores of the donor/acceptor type have been designed, synthe-
sized, and characterized. Their TPA cross sections are high in the
near-IR range with σ2 of 470 GM at 810 nm for the diphenyl-
amino-substituted chromophore 6a and 200ꢀ300 GM at
700 nm for the chromophores 6b and 6c containing oxygen
donor groups. The inclusion of the p-carborane entity into TPA
chromophores brings 10 boron atoms in the structure and does
not reduce the TPA cross sections σ2 of the whole conjugated
systems. Two of the new chromophores (6a and 6c) are
sufficiently water-soluble for use in TPEM in living cells. The
high σ2 of 6a and its high boron atom content make it a
bifunctional molecule that could be of great interest in biology,
to study by TPEM the mechanisms involved in BNCT. Further
work is in progress to test it in two-photon bioimaging, and
cellular results will be reported in a forthcoming paper.
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: jean-francois.nicoud@unistra.fr. Tel: þ33 (0)3 68 85 42 76.
Fax: þ33 (0)3 68 85 43 13.
’ ACKNOWLEDGMENT
CNRS and Universitꢀe de Strasbourg are gratefully acknowl-
edged for financial support, and A.H. is grateful to the French
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ministry of research for a fellowship. Dr. Pascal Kesler is
acknowledged for assistance in microscopy. Dr. Etienne Piꢀemont
is acknowledged for lifetime measurements.
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