Intensely fluorescent azobenzenes: Synthesis, crystal structures, effects of substituents, and application to fluorescent vital stain

Article abstract of DOI:10.1002/chem.201000258

2-[Bis(pentafluorophenyl)boryl]azobenzenes bearing hydrogen, methoxy, dimethylamino, trifluoromethyl, fluoro, n-butyl, and tert-butyldimethylsiloxy groups at the 4′-position or methoxy and bromo groups at the 4position have been synthesized. The 4bromo group of the 2-boryl-4-bromoazobenzene derivative was converted to phenyl and diphenylamino groups by palladium-catalyzed reactions. The absorption and fluorescence properties have been investigated using UV/Vis and fluorescence spectroscopy. The 2-borylazobenzenes emitted an intense green, yellow, and orange fluorescence, in marked contrast to the usual azobenzene fluorescence. The 4′-siloxy derivative showed the highest fluorescence quantum yield (0.90) among those reported for azobenzenes to date. The correlation between the substituent and the fluorescence properties was elucidated by studying the effect of the substituent on the relaxation process and from DFT and TD-DFT calculations. An electron-donating group at the 4′-position was found to be important for an intense emission. Application of fluorescent azobenzenes as a fluorescent vital stain for the visualization of living tissues was also investigated by microinjection into Xenopus embryos, suggesting these compounds are nontoxic towards embryos.

Full text of DOI:10.1002/chem.201000258

DOI: 10.1002/chem.201000258
Intensely Fluorescent Azobenzenes: Synthesis, Crystal Structures, Effects of
Substituents, and Application to Fluorescent Vital Stain
Junro Yoshino,[a] Akiko Furuta,[a] Tetsuya Kambe,[a] Hiroaki Itoi,[a] Naokazu Kano,*[a]
Takayuki Kawashima,*[a] Yuzuru Ito,[b] and Makoto Asashima*[b, c]
Abstract:
2-[Bis(pentafluorophenyl)-
borylazobenzenes emitted an intense
green, yellow, and orange fluorescence,
in marked contrast to the usual azo-
benzene fluorescence. The 4’-siloxy de-
rivative showed the highest fluores-
cence quantum yield (0.90) among
those reported for azobenzenes to date.
The correlation between the substitu-
ent and the fluorescence properties
was elucidated by studying the effect of
the substituent on the relaxation pro-
cess and from DFT and TD-DFT calcu-
lations. An electron-donating group at
the 4’-position was found to be impor-
tant for an intense emission. Applica-
tion of fluorescent azobenzenes as a
fluorescent vital stain for the visualiza-
tion of living tissues was also investi-
gated by microinjection into Xenopus
embryos, suggesting these compounds
are nontoxic towards embryos.
boryl]azobenzenes bearing hydrogen,
methoxy, dimethylamino, trifluoro-
methyl, fluoro, n-butyl, and tert-butyl-
dimethylsiloxy groups at the 4’-position
or methoxy and bromo groups at the 4-
position have been synthesized. The 4-
bromo group of the 2-boryl-4-bromo-
ACHTUNGTRENNUNGazobenzene derivative was converted
to phenyl and diphenylamino groups
by palladium-catalyzed reactions. The
absorption and fluorescence properties
have been investigated using UV/Vis
and fluorescence spectroscopy. The 2-
Keywords: azo compounds
ranes · fluorescence · fluorescent
probes · substituent effects
· bo-
AHCTUNGTRENNUNG
Introduction
as photoresponsive molecular switches, by taking advantage
of their photoisomerization.[2] However, their characteristic
photoisomerization inhibits another important property of
coloring materials, that is, fluorescence. The photoisomeriza-
tion process is so efficient in photoexcited azobenzenes that
their fluorescence emission process, which is slower, is not
competitive.[3] Hence, azobenzenes themselves virtually do
not emit light.[4] The fluorescence quantum yield of an un-
substituted azobenzene has been reported to be FF =2.53ꢀ
10À5.[5] There have been only limited examples of azoben-
zene derivatives that show fluorescence. Some azobenzene
derivatives in a rigid matrix at low temperature,[6] ortho-
metalated azobenzenes,[7] and self-assembled aggregates of
azobenzene derivatives[8] have been reported to emit fluo-
rescence. However, most of their fluorescence quantum
yields at ambient temperature in solution are about FF =
10À3, which is still low compared with fluorescent organic
materials. In general, azobenzenes remain as nonfluorescent
coloring materials, while many other organic dye molecules,
such as rhodamines and fluoresceins, have been developed
as highly emissive fluorescent materials, and have been uti-
lized for the labeling of biomolecules, such as proteins, to
make them detectable.[9] Boron-containing fluorescent mate-
rials, for example, boron dipyrromethenes and boron diketo-
Azobenzene is a common coloring material, especially for
dyes, used around the world.[1] Azobenzenes are also used
[a] Dr. J. Yoshino, A. Furuta, T. Kambe, H. Itoi, Prof. N. Kano,
Prof. T. Kawashima
Department of Chemistry, Graduate School of Science
The University of Tokyo, 7-3-1 Hongo
Bunkyo-ku, Tokyo 113-0033 (Japan)
Fax : (+81)3-5800-6899
E-mail: kano@chem.s.u-tokyo.ac.jp
takayuki@chem.s.u-tokyo.ac.jp
[b] Dr. Y. Ito, Prof. M. Asashima
Organ Development Research Laboratory
National Institute of Advanced Industrial Science and
Technology (AIST)
Tsukuba Central 4, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562 (Japan)
[c] Prof. M. Asashima
Department of Life Science (Biology)
Graduate School of Arts and Sciences
The University of Tokyo, 3-8-1 Komaba
Meguro-ku, Tokyo 153-8902 (Japan)
Fax : (+81)298612987
E-mail: asashi@bio.c.u-tokyo.ac.jp
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201000258.
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FULL PAPER
nates, have also been applied in biological imaging.[10] If azo-
benzenes could fluoresce, then they would be useful as fluo-
rescent materials applicable to light-emitting devices, fluo-
rescent probes, and molecular detection, because of their
easy synthesis and high modification capability.
4-position (R’), respectively, were synthesized in a similar
manner. The relatively inactive nature of the bis(pentafluo-
ACTHNGUTRENNUG ACHNTUGRENpNUG henyl)boranes, which are stabilized by a strongly coordi-
A
ACHTUNGTRENNUNG
We recently reported on the synthesis of intensely fluores-
cent boron-substituted azobenzenes[11,12] in the continuation
of our work on 2-borylazobenzene derivatives[13,14] by utiliz-
N
ACHTUNGTRENNUNG
À
ing a B N interaction, which is a tool to adjust the structure
and properties of p-conjugated molecules.[15] To make these
fluorescent azobenzenes useful as a fluorescent organic ma-
terial, the fundamental relationship between their molecular
structure and their fluorescence properties needs to be eluci-
dated, and their properties should be predictable from their
structure. In addition, their nontoxicity to living cells needs
to be confirmed. Here, we report on synthetic methods of
fluorescent 2-borylazobenzenes and their fluorescence prop-
erties in detail, as well as the correlation between the sub-
stituent and the fluorescence properties. We also report on
their application to the visualization of living cells to check
on their nontoxicity.
Results
Synthesis: 2-Borylazobenzenes 1a–g bearing hydrogen,[11]
methoxy,[11] dimethylamino,[12] trifluoromethyl, fluoro, n-
butyl, and tert-butyldimethylsiloxy groups at the 4’-position
(R), respectively, were synthesized from the corresponding
iodoazobenzenes 2a–g, n-butyllithium, and fluoroborane 3a
or ethoxyborane 3b (Scheme 1 and Table 1). 2-Borylazoben-
zenes 1h and 1i bearing methoxy and bromo groups at the
Scheme 2. Suzuki–Miyaura cross-coupling reaction of 1i.
toluene in the presence of excess sodium tert-butoxide,
30 mol% of palladium(II) acetate, and 60 mol% of 1,1’-bis-
AHCTUNGTREG(NNUN diphenylphosphino)ferrocene (DPPF) gave 2-boryl-4-(di-
phenylamino)azobenzene 1k (58%; Scheme 3).
Scheme 1. Synthesis of 2-borylazobenzenes 1a–i.
Table 1. Yields of 1a–i.
Scheme 3. Buchwald–Hartwig amination reaction of 1i.
R
R’
Yield [%]
A 2-(boryloxy)azobenzene derivative was also synthesized
to investigate the effect of the direct binding between the
boron atom and azobenzene moiety on the fluorescence of
azobenzene. 2-Boryloxyazobenzene 5, a six-membered-ring
analogue of 1a, was synthesized by condensation of ethoxy-
borane 3b and 2-hydroxyazobenzene (4), which was conve-
1a
1b
1c
1d
1e
1 f
1g
1h
1i
H
OMe
NMe2
CF3
F
nBu
OSitBuMe2
H
H
H
H
H
H
H
H
H
OMe
Br
28[a]
27[a]
7[b]
12[b]
36[b]
47[b]
42[b]
28[b]
26[b]
AHCTUNGTRENNUNG
A
ACHTUNGTRENNUNG
[a] Synthesized from fluoroborane 3a. [b] Synthesized from ethoxyborane
3b.
1
and boryloxyazobenzene 5 were characterized using H, 11B,
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N. Kano, T. Kawashima, M. Asashima et al.
Table 2. Absorption maxima (labs), emission maxima (lem), colors, Stokes
shifts, and fluorescence quantum yields (FF) of 1a–k and 5 in hexane at
RT.
labs
Solution
color
lem
Emission
color
Stokes
FF
[nm]
[nm]
shift [cmÀ1
]
1a
1b
1c
1d
1e
1 f
1g
1h
1i
386
439
516
382
390
402
433
443
416
445
553
474
yellow
yellow
pink
503
524
566
511
504
507
524
538
509
539
634
576
green
6000
3700
1700
6600
5800
5200
4000
4000
4400
3900
2300
3700
0.20
0.76
0.52
0.039
0.29
0.31
0.90
0.048
0.045
0.13
0.37
0.033
yellow–green
orange
green
green
green
yellow–green
yellow
yellow
yellow
red
Scheme 4. Synthesis of 2-(boryloxy)azobenzene 5.
yellow
yellow
yellow
yellow
yellow
orange
orange
purple
orange
13C, and 19F NMR spectroscopy, mass spectrometry, and ele-
mental analysis. The tetracoordination state of their boron
atoms was confirmed from the 11B NMR chemical shifts (dB
À0.4 to À3.2) in CDCl3.
1j
1k
5
orange
X-ray crystallographic analysis: X-ray crystallographic anal-
ysis was performed for 1a, 1b, and 1d. In all cases, there
À
was an intramolecular B N interaction to form tetracoordi-
nate boron atoms. The crystal structures of 1a, 1b, and 1d
the azobenzenes was observed between 503 and 634 nm, as
shown in Table 2 and Figure 4.
À
are shown in Figure 1. The bond lengths of the B N dative
bonds (1a: 1.625(2), 1b: 1.633(2), and 1d: 1.638(3) ꢂ) and
the N=N bonds (1a: 1.2704(18), 1b: 1.2798(17), and 1d:
1.276(2) ꢂ) are not significantly different from each other,
despite the large electronic difference in the substituent at
the 4’-position. In 1b, the bond lengths of the benzene ring
attached to the N2 atom are different from the bond lengths
of 1a[11] due to the contribution of a quinoid structure,
Fluorescence properties in the solid state: Azobenzenes 1a,
1b, 1d, 1e, and 1h showed fluorescence in the solid state
under UV irradiation, whereas azobenzene 1c did not show
any fluorescence at all. The strong donor–acceptor character
of 1c may make its intermolecular interaction strong
enough to quench its fluorescence in the solid state. In the
fluorescence spectra in the solid state, 1a, 1b, 1d, 1e, and
1h showed an emission maximum in the wavelength region
between 504 and 588 nm (Table 3 and Figure 5).
À
À
judged from the C C bond lengths (C7 C8: 1.395(2),
À
À
À
C8 C9: 1.384(2), C9 C10: 1.397(2) ꢂ), the C7 N2 bond
À
length (1.4191(19) ꢂ), and the C10 O1 bond length
(1.3558(19) ꢂ).
Microinjection into Xenopus embryos: To examine whether
microinjection of a fluorescent azobenzene could be used as
a vital fluorescent tracer, we diluted a solution of fluores-
cent azobenzene 1b in dimethyl sulfoxide (DMSO) with
water, and injected it into dorsal/animal blastomeres (pro-
spective central nervous system region) at the eight-cell
stage of Xenopus embryos (Figure 6A), and observed the
phenotypes. Xenopus embryos are fertilized externally and
all stages of embryo development are easily accessible,
making it relatively easy to study a fluorescent tracer experi-
ment by microinjection into embryonic cells. As a control
Absorption and fluorescence properties in solution: In the
UV/Vis spectra in hexane, the azobenzenes 1a–k and 5
showed an absorption maxima in the wavelength region be-
tween 382 and 553 nm, as shown in Table 2 and Figure 2.
Azobenzenes 1a–k and 5 showed green, yellow, orange, or
red fluorescence in hexane at room temperature (RT) upon
irradiation with a UV lamp (l=360 nm), in contrast to that
usually observed for azobenzenes (Figure 3). In the fluores-
cence spectra in hexane at RT, the emission maximum of
Figure 1. ORTEP drawings of A) 1a, B) 1b, and C) 1d with a thermal ellipsoid plot (50% probability).
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Fluorescent Azobenzenes
FULL PAPER
Figure 2. Absorption spectra of 1a–k and 5 in hexane.
Figure 4. Fluorescence spectra of 1a–k and 5 in hexane.
experiment, GFP mRNA was also injected into other em-
bryos at the same stage (Figure 6B and 6C). The embryos
were illuminated with excitation light at a wavelength of
488 nm and a yellow–green fluorescence was detected just
after microinjection (data not shown). Fluorescence decay
was not detected at the neurula stage (24 h after injection),
tailbud stage (48 h after injection), and tadpole stage (120 h
after injection) (Figure 6D–F). The fluorescence intensity at
each stage was slightly weaker than that of control embryos
injected with GFP mRNA. There was no difference in the
above results between using DMSO or ethanol as the sol-
vent (See Supporting Information, Figure S1). In contrast to
conventional aqueous fluores-
cent dyes, the embryos injected
with the fluorescent azoben-
zene showed a punctate image
with their development (Fig-
ure 6F and Figure S1C in the
Supporting Information).
Figure 3. Photographs of 1a–g and 5 (left to right) in hexane under UV irradiation (l=360 nm).
Chem. Eur. J. 2010, 16, 5026 – 5035
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N. Kano, T. Kawashima, M. Asashima et al.
Table 3. Emission maxima (lem), maxima of the excitation spectra (lmax),
and Stokes shifts of 1a–e and 1h in the solid state at RT.
lem
Emission
color
lmax
Stokes
[nm]
[nm]
shift[a] [cmÀ1
]
1a
1b
1c
1d
1e
1h
510
588
–
518
504
559
green
red
–
yellow–green
green
orange
457
578
2300
300
[b]
[b]
456
456
538
2600
2100
900
[a] The Stokes shift was calculated by estimating that lmax was equal to
the absorption maximum. [b] No fluorescence was observed.
Figure 5. Fluorescence spectra of 1a, 1b, 1d, 1e, and 1h in the solid
state.
Discussion
Effect of the substituent on the absorption and fluorescence
properties: In azobenzenes 1a–g, the stronger electron-do-
nating group at the 4’-position gave a larger red shift of the
p–p* absorption maximum and a larger Stokes shift. Both
the red shift of the absorption maximum and the large
Stokes shift resulted in a red shift of the emission maximum
of the azobenzenes bearing an electron-donating group at
the 4’-position. The observed tendency on the Stokes shift
indicates that the stronger electron-donating group at the 4’-
position more strongly suppresses structural changes upon
excitation. The stronger electron-donating group also leads
to a higher fluorescence quantum yield, with the exception
of 1c. In particular, the fluorescence quantum yield of 1g
was determined as FF =0.90, which was more than 30000
times higher than that of the unsubstituted azobenzene (7).
In our previous report, we reported that 1b (FF =0.76) was
the most intensely fluorescent azobenzene to date.[11] Now,
azobenzene 1g, bearing a stronger electron-donating siloxy
group at the 4’-position, is the most intensely fluorescent
azobenzene. In 1e, the fluoro group at the 4’-position is not
considered to work as an electron-withdrawing group, but as
an electron-donating group due to its lone pair conjugating
with the p orbital of the azo group, judging from the charac-
teristics of the absorption and emission wavelengths shown
in Table 2. Compound 1h, with a 4-methoxy group, showed
an absorption wavelength and Stokes shift similar to those
of 1b with a 4’-methoxy group. These results suggest that
the absorption wavelength and the Stokes shift are mainly
determined by the electron-donating ability of substituent
Figure 6. Fluorescence images of Xenopus embryos: A) A schematic il-
lustration of the microinjection process. Eight-cell-stage Xenopus em-
bryos were bilaterally microinjected with GFP mRNA or fluorescent
azobenzene 1b. B) and C) GFP mRNA injected embryos. At the neurula
(B) and tailbud (C) stages, a green fluorescence was detected. D)–F) Flu-
orescent azobenzene 1b was dissolved in DMSO. At the neurula (D),
tailbud (E), and tadpole (F) stages, a yellow-green fluorescence was de-
tected (shown by the arrowhead). The bright-field image is on the left-
hand side and the dark-field image is on the right-hand side. Intrinsic
fluorescence was detected in the endodermal region (shown by the aster-
isk). The scale bars denote a distance of 0.5 mm.
on the azobenzene moiety, and are almost independent of
their position. In contrast, the fluorescence quantum yield
of 1h was much lower than that of 1b, and even that of 1a.
The position of the substituent on the azobenzene moiety
was found to be an important factor influencing the fluores-
cence quantum yield in this case. 2-Boryloxyazobenzene 5
achieved a higher red-shifted emission than the correspond-
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Fluorescent Azobenzenes
FULL PAPER
ing 2-borylazobenzene 1a, while its fluorescence quantum
yield was much lower. The presence of an oxygen atom and
the absence of a boron atom at the ortho-position to the azo
group in the azobenzene moiety in 5 makes the fluorescence
properties so different from those of 1a–k.
Effect of the substituent on the relaxation process: The fluo-
rescence lifetimes of 1a–d, 1g, 1h, and 5 in hexane were
measured, as shown in Table 4. The rate constants of the ra-
Table 4. Fluorescence lifetimes (tF) and calculated rate constants of the
radiative (kr) and nonradiative (knr) relaxation of 1a–d, 1g, 1h, and 5 in
hexane at RT.
[b]
tF [ns]
3.82(4)
6.24(5)
6.17(5)
2.67(3)
5.50(2)
kr[a] [sÀ1
]
knr [sÀ1
]
1a
1b
1c
1d
1g
1h
5
5.2ꢀ107
12ꢀ107
21ꢀ107
3.8ꢀ107
7.8ꢀ107
36ꢀ107
8.4ꢀ107
1.5ꢀ107
16ꢀ107
1.8ꢀ107
>95ꢀ107
23ꢀ107
<1
4.29(4)
>4.8ꢀ107
0.77ꢀ107
[a] kr =FF/tF. [b] knr =k
C
diative and nonradiative relaxation (kr and knr, respectively)
of the excited state were calculated from their lifetime (tF)
and quantum yields (FF) by using the equation, FF =
kr/ACHTUNGTRENNUNG
(kr+knr)=krtF,[16] to evaluate the effect of the substituent
on the fluorescence quantum yield and relaxation processes
of their excited states (Table 4). The radiative rate constant
(kr) of 1a (kr =5.2ꢀ107 sÀ1) was higher than that of unsubsti-
tuted azobenzene 6 (kr =1.5ꢀ106 sÀ1),[5] whereas the nonra-
diative rate constant (knr) of 1a (knr =2.1ꢀ108 sÀ1) was lower
than that of 6 (knr =2.0ꢀ1012 sÀ1).[5] Both the high value of kr
and the low value of knr will lead to the higher fluorescence
efficiency of 1a. An increase in the rate constant kr reflects
an enhancement of the radiative relaxation from the singlet
excited state. A decrease in the rate constant knr is caused
by the suppression of the nonradiative relaxation from the
singlet excited state through processes such as an internal
conversion and photoisomerization. Therefore, introduction
of the bis(pentafluorophenyl)boryl group to azobenzene at
the 2-position should enhance the radiative relaxation pro-
cess of azobenzene by changing its electronic state, and sup-
pressing any photoisomerization process around the N=N
double bond, which is responsible for the highly efficient ra-
diationless deactivation in most azobenzenes. This suppres-
sion is considered to be provided by the rigid structure of
Figure 7. A) Calculated energy levels of the frontier orbitals of (E)-azo-
benzene (6), 1a–e, and 1h at the B3PW91/6-31+G(d)//B3PW91/6-
31G(d) level of theory. The orbitals denoted by blue and red levels origi-
nate from the p (p*) and n orbitals of the azo group, respectively. B) Cal-
culated singlet excitation energies of (E)-azobenzene (6), 1a–e, and 1h
using the TD-DFT method at the B3PW91/6-31+G(d)//B3PW91/6-
31G(d) level of theory. The excited states denoted by the blue and red
1(p,p*) and 1(n,p*) states, respectively.
levels have the character of ACTHUNGTRENNUNG HCATUNGTRENNUNG
forded an inversion of the order of the
N
ACHTUNGTRENNUNG
1(p,p*)
states on the energy scale. As a result, the ACHTUNGTRENNNUG
state of 1a becomes the lowest singlet excited state (S1),
providing an increase in the transition probability from the
S1 state to the ground state based on the orbital symmetries
(Figure 7B).
Next, the effect of the substituents at the 4- and 4’-posi-
tions on the electronic state and excitation energy was inves-
tigated. In 1b, 1c, 1e, and 1h, the same results were ob-
tained from the DFT and TD-DFT calculations as shown in
À
1a around the azo group, because of the tight B N interac-
tion between the boron and the nitrogen atoms. The elec-
tronic state and excitation energy of 1a and 6 were investi-
gated to reveal the effect of the boryl group substituent on
the electronic state of azobenzene.[11] DFT and TD-DFT cal-
culations at the B3PW91/6-31+G(d)//B3PW91/6-31G(d)
Figure 7. In 1d, although the
ACHTUNGTRENNUNG
the S1 state, the energy levels of the AHCUTNGTRENNUNG
and the S1 state were close to each other. Therefore, this sit-
uation explains the emissive nature with a low fluorescence
quantum yield. A transition from the S1 state to the ground
state is normally forbidden. However, there is a probability
À
level of theory revealed that the B N bonding interaction
stabilizes the n orbital of the azo group, as shown in Fig-
ure 7A. The lowering of the energy level of the n orbital af-
1(p,p*) excited state changing to the S1 state, provid-
of the ACHTNUGRTENNUGN
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N. Kano, T. Kawashima, M. Asashima et al.
ing an increase in the transition probability from the S1 state
to the ground state through an interaction between a mole-
cule of 1d and a surrounding molecule, such as a solvent
molecule.
Conclusion
2-Borylazobenzenes bearing various substituents at the 4-
and 4’-positions were synthesized. The transformation of a
substituent was achieved from the Suzuki–Miyaura cross-
coupling reaction and the Buchwald–Hartwig amination re-
action, even in the presence of a bis(pentafluorophenyl)bo-
In 1a, 1b, 1d, and 1g, the stronger electron-donating
groups at the 4’-position accelerate the radiative relaxation
and slow down the nonradiative relaxation of their excited
states. The high rate of the radiative relaxation would be
caused by the high oscillator strength of the transition be-
tween the lowest singlet excited state and the ground state
due to the donor–p-acceptor molecular structure. The low
rate of the nonradiative relaxation would be caused by the
enhanced rigidity of the molecular structure induced by the
delocalization of the lone pair of the donor group onto the
p-conjugated system of the azo group, which appears as the
difference in the degree of contribution of the quinoid struc-
ture between 1a and 1b in their crystal structures. In 1h,
the methoxy group at the 4-position gave a very high nonra-
diative relaxation rate, which was responsible for its low
fluorescence quantum yield. The reason for the high nonra-
diative relaxation rate is unclear, but some nonradiative re-
laxation processes, such as a relaxation through triplet states
formed by intersystem crossings, may be involved.
AHCTUNGTREGrNNNU yl group at the 2-position of the azobenzene. These com-
pounds showed yellow, green, orange, and red fluorescence,
which was extremely intense for azobenzene derivatives.
Based on observations of the detailed fluorescence proper-
ties of 2-borylazobenzene derivatives and the effect of their
substituents, a correlation between the substituent and the
fluorescence properties was elucidated, and this is consid-
ered to make their fluorescent properties more predictable.
Microinjection into Xenopus embryos revealed a fluores-
cence emission, even after staining, and this suggests its non-
toxicity to living cells, which is important for application as
a fluorescent vital stain and a fluorescent tracer in the
future.
Experimental Section
General: Solvents were purified before use by reported methods. All re-
actions were carried out under argon atmosphere unless otherwise noted.
The 1H NMR (400 MHz), 11B NMR (128 MHz), 13C NMR (100 MHz),
and 19F NMR (376 MHz) spectra were measured with a JEOL AL400
spectrometer. The 13C NMR (126 MHz) spectra were measured with a
Bruker DRX-500 spectrometer. Tetramethylsilane was used as an exter-
nal standard for the 1H and 13C{1H} NMR spectra. BF3·OEt2 and CCl3F
were used as external standards for the 11B and 19F NMR spectra, respec-
tively. Low- and high-resolution mass spectra were measured with a Shi-
madzu GCMS-QP2010 gas chromatograph mass spectrometer and JEOL
MStation JMS-700 spectrometer, respectively. Absorption spectra were
measured with a Hitachi U-3500 UV/Vis spectrometer and JASCO V-530
and V-670 UV/Vis spectrometers. Fluorescence spectra were measured
with a JASCO FP-6500 fluorescence spectrophotometer. Preparative gel
permeation liquid chromatography was performed by LC-908 and LC-
918 with JAIGEL H1+H2 columns (Japan Analytical Industry) with tol-
uene or chloroform as the eluent. All melting points were uncorrected.
Elemental analyses were performed by the Microanalytical Laboratory
of Department of Chemistry, Faculty of Science, the University of Tokyo.
Application to fluorescent vital stain: Azobenzenes have
been used as dyes for a long time, and are considered to be
nonfluorescent materials. However, we have succeeded in
making them highly fluorescent by taking advantage of the
À
N B interaction, as described above. One conceivable appli-
cation of fluorescent azobenzenes in the future is as a fluo-
rescent vital stain for chemical biology. Nontoxicity to living
cells and intense fluorescence, even after staining, are essen-
tial requirements for their application as a vital fluorescent
tracer. Importantly, the microinjection of 1b into Xenopus
embryos did not induce any malformation. This is a signifi-
cant result in that fluorescent azobenzene was nontoxic for
cells, and did not inhibit the normal development of Xeno-
pus embryos. Furthermore, a yellow–green fluorescence was
detected from the embryos, and a marked fluorescence
decay was not detected (Figure 6D–F and Figure S1 in the
Supporting Information), although the intensity of the fluo-
rescence was slightly weaker than that of the control em-
bryos injected with GFP mRNA (Figure 6B and 6C). The
perceptible fluorescence after both injection and develop-
ment suggests its availability as a fluorescent tracer. The
punctate image of the fluorescent azobenzene in the em-
bryos, along with their development, suggests that crystalli-
zation of the injected azobenzene proceeded during embryo-
genesis (Figure 6F and S1C).
2-Iodo-4’-(trifluoromethyl)azobenzene (2d),[14] 4-fluoro-2’-iodo
zene (2e),[14] 4-butyl-2’-iodoazobenzene (2 f),[17] 4-hydroxy-2’-iodo
zene (2g’),[11] 2-iodo-4-methoxyazobenzene (2h)[18] and ethoxybis
A
ACHTUNGTRENNUNG
A
R
ACHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
fluorophenyl)borane (3b)[19] were prepared according to the literature.
Preparation of 2-hydroxyazobenzene (4)[20] is described in the Supporting
Information. The fluorescence quantum yields were measured with a Ha-
mamatsu absolute photo-luminescence quantum yield measurement
system C9920–02.
Synthesis of 4-(tert-butyldimethylsiloxy)-2’-iodoazobenzene (2g): A solu-
tion of 2g’ (417 mg, 1.29 mmol), imidazole (281 mg, 4.08 mmol), and tert-
butylchlorodimethylsilane (259 mg, 1.72 mmol) in dichloromethane
(20 mL) was stirred at RT for 27 h. The reaction mixture was treated
with aqueous ammonium chloride (30 mL) and extracted with chloro-
form (10 mLꢀ3). After the organic layer was dried over anhydrous mag-
nesium sulfate, evaporation of the solvent and separation by silica gel
column chromatography (hexane/chloroform) gave 2g (535 mg, 95%).
One unsolved problem is the technical difficulty in the in-
jection of thick fluorescent azobenzene due to its low solu-
bility in water. If we could develop a highly water-soluble
fluorescent azobenzene, then injected embryos would show
significantly higher fluorescence intensity.
1
Dark red liquid; H NMR (400 MHz, CDCl3, TMS): d=0.26 (s, 6H), 1.01
(s, 9H), 6.96 (d, 3J(H,H)=8.8 Hz, 2H), 7.12 (t, 3J
ACTHNUGTRENNUGN ACHTUNGTRENNNUG
7.40 (t, 3J
N
(H,H)=7.6 Hz, 1H), 7.60 (d, 3J
ACHUTGTNRENNUG CAHTUNGTRENNUNG
3
3J
5032
www.chemeurj.org
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 5026 – 5035

Fluorescent Azobenzenes
FULL PAPER
117.85 (s), 121.117 (s), 125.98 (s), 129.34 (s), 132.16 (s), 140.23 (s), 147.75
(s), 151.83 (s), 159.68 ppm (s); MS (EI, 70 eV): m/z (%): 438 (65) [M+],
381 (19), 235 (32), 207 (100), 203 (58), 150 (77), 135 (56), 73 (58); HRMS
(FAB+): m/z calcd for C18H24IN2OSi [M+H]+: 439.0703; found:
439.0623.
(s, quaternary C); a signal due to an aromatic carbon could not be detect-
ed; 19F NMR (376 MHz, CDCl3, CCl3F): d=À162.32 to À162.11 (m, 4F),
À155.89 (t, 3J
(F,F)=20 Hz, 2F), À131.08 to À130.90 ppm (m, 4F); UV/
N
Vis (hexane) lmax (e)=247 (1.3ꢀ104), 402 nm (2.2ꢀ104 dm3 molÀ1 cmÀ1);
MS (EI, 70 eV): m/z (%): 582 (100) [M+], 563 (13), 540 (64), 282 (24),
134 (41), 91 (75); HRMS (FAB+): m/z calcd for C28H17BF10N2: 582.1325;
found 582.1347.
Synthesis of bis(pentafluorophenyl){2-[4-(trifluoromethyl)phenylazo]-
A
ACHTUNGTRENNUNG
a
Synthesis of {2-[4-(tert-butyldimethylsiloxy)phenylazo]phenyl}bis(penta-
fluorophenyl)borane (1g): A similar reaction to that described for the
preparation for 1d by using 2g (1.10 g, 2.51 mmol) in Et2O (300 mL)
with nBuLi (1.53m in hexane, 2.97 mL, 3.01 mmol) and 3b (0.78 mL,
3.0 mmol) gave 1g (694 mg, 42%). Yellow crystals (MeOH); m.p. 138.9–
139.68C; 1H NMR (400 MHz, CDCl3, TMS): d=0.26 (s, 6H), 0.98 (s,
À1128C. The reaction solution was stirred for 30 min and then added to
a solution of 3b (0.90 mL, 3.4 mmol) in Et2O (20 mL) at À1128C. After
the solution was allowed to warm gradually to room temperature and the
solvent was removed in vacuo, the residue was dissolved in chloroform
and insoluble materials were removed by filtration. Concentration of the
filtrate followed by separation with gel permeation chromatography
(eluent: toluene) afforded 1d (222 mg, 12%). Yellow brown crystals
9H), 6.86 (d, 3J
A
3J
3J
C
ACHTUNGTRENNUNG
1
(CHCl3/hexane); m.p. 212.8–213.58C; H NMR (400 MHz, CDCl3, TMS):
À0.5 ppm (line width h1/2 =291 Hz); 13C{1H} NMR (100 MHz, CDCl3,
TMS); d=À4.22 (s; CH), 18.24 (s; CH), 25.53 (s; CH), 114.82 (brs; CB),
120.79 (s; CH), 124.60 (s; CH), 127.71 (s; CH), 128.22 (s; CH), 128.36 (s;
CH), 134.90 (s; CH), 135.91–138.32 (m; CF), 138.07 (s; CH), 138.38–
141.23 (m; CF), 146.33–149.23 (m; CF), 154.21 (brs; CB), 156.15 (s),
160.12 (s; CO); 19F NMR (376 MHz, CDCl3, CCl3F): d=À162.35 to
d=7.54–7.59 (m, 1H), 7.62–7.67 (m, 1H), 7.67–7.71 (m, 1H), 7.73–7.78
(m, 2H), 8.14–8.19 (m, 2H), 8.40 ppm (d, 3J
ACTHNUTRGNE(NUG H,H)=7.8 Hz, 1H);
11B NMR (128 MHz, CDCl3, BF3·OEt2): d=À0.5 ppm (line width h1/2
=
179 Hz); 13C{1H} NMR (100 MHz, CDCl3, TMS): d=113.66 (brs; CB),
122.99 (s; CH), 123.24 (q, 1J
3.8 Hz; CH), 128.75 (s; CH), 129.04 (s; CH), 129.88 (s; CH), 133.86 (q,
2J
(C,F)=34 Hz; CCF3), 135.87–138.85 (m; CF), 137.30 (s; CH), 138.90–
(C,F)=273 Hz; CF3), 127.03 (q, 3J
ACHUTGTNRNEUNG AHCUTNGTREN(NUGN C,F)=
ACHTUNGTRENNUNG
À162.22 (m, 4F), À155.99 (t, 3J
(F,F)=20 Hz, 2F), À131.16 to
A
141.94 (m; CF), 145.80 (s; CN), 146.37–149.22 (m; CF), 156.66 (s; CN),
156.73 ppm (brs; CB); 19F NMR (376 MHz, CDCl3, CCl3F): d=À163.35
À131.08 ppm (m, 4F); UV/Vis (hexane) lmax (e)=250 (8.6ꢀ103), 433 nm
(1.6ꢀ104 dm3 molÀ1 cmÀ1); MS (FAB+): m/z: 656 [M+]; elemental analysis
calcd (%) for C30H23BF10N2OSi: C 54.89, H 3.58, N, 4.27; found: C 54.72,
H 3.80, N, 4.25.
to À163.16 (m, 4F), À156.60 (t, 3J
(F,F)=19 Hz, 2F), À132.62 (d,
E
3J
(F,F)=24 Hz, 4F), À65.20 ppm (s, 3F); UV/Vis (hexane): lmax (e)=
382 nm (2.2ꢀ104 dm3 molÀ1 cmÀ1); MS (EI, 70 eV): m/z (%): 594 (100)
[M+], 575 (19), 145 (41); elemental analysis calcd (%) for C25H8BF13N2:
C 50.54, H 1.36, N 4.71; found: C 50.80, H 1.54, N 4.67.
Synthesis of [4-methoxy-2-(phenylazo)phenyl]bis(pentafluorophenyl)bo-
AHCTUNGERTGrNNUN ane (1h): A similar reaction to that described for the preparation for 1d
by using 2h (1.35 g, 4.00 mmol) in Et2O (80 mL) with nBuLi (1.6m in
hexane, 2.75 mL, 4.40 mmol) and 3b (1.25 mL, 4.74 mmol) in Et2O
(20 mL) gave 1h (629 mg, 28%). Orange crystals (hexane); m.p. 161.0–
161.98C (decomp); 1H NMR (400 MHz, CDCl3, TMS): d=3.91 (s, 3H),
Synthesis of [2-(4-fluorophenylazo)phenyl]bis(pentafluorophenyl)borane
(1e): A similar reaction to that described for the preparation for 1d by
using 2e (596 mg, 1.83 mmol) in Et2O (40 mL) with nBuLi (1.6m in
hexane, 1.3 mL, 2.1 mmol) and a solution of 3b (0.90 mL, 3.4 mmol) in
Et2O (20 mL) gave 1e (354 mg, 36%). Brown crystals (hexane); m.p.
160.4–163.88C (decomp); 1H NMR (400 MHz, CDCl3, TMS): d=7.13–
6.99 (dd, 3J
2.4 Hz, 1H), 7.39–7.45 (m, 3H), 7.88–7.95 (m, 2H), 8.24 ppm (d,
3J(H,H)=8.8 Hz, 1H); 11B NMR (128 MHz, CDCl3, BF3·OEt2): d=
(H,H)=8.8 Hz, 4J(H,H)=2.4 Hz, 1H), 7.11 (d, 4J
ACHTUNGTRENGNUN ACHTUNRTGENNUNG ACHTUNGTRENNUNG(H,H)=
AHCTUNGTRENNUNG
7.20 (m, 2H), 7.50–7.55 (m, 1H), 7.55–7.61 (m, 1H), 7.64 (d, 3J
ACHTUNGTRENNUNG
À1.8 ppm (line width h1/2 =117 Hz); 13C{1H} NMR (100 MHz, CDCl3,
TMS): d=56.12 (s; CH3), 113.08 (s; CH), 114.55 (brs; CB), 115.55 (s;
CH), 121.72 (s; CH), 129.48 (s; CH), 131.17 (s; CH), 131.22 (s; CH),
135.57–141.68 (m; CF, two signals overlapped each other), 143.70 (s; qua-
ternary C), 146.17–149.21 (m; CF), 151.23 (s; quaternary C), 160.34 (brs;
7.2 Hz, 1H), 8.03–8.09 (m, 2H), 8.31 ppm (d, 3J
ACHTUNGTRENNUNG
11B NMR (128 MHz, CDCl3, BF3·OEt2): d=À0.4 ppm (line width h1/2
=
183 Hz); 13C{1H} NMR (100 MHz, CDCl3, TMS): d=113.91 (brs; CB),
117.06 (d, 2J
ACHTUNGTRENNUNG ACHTUNGTRNE(NUGN C,F)=9.6 Hz; CH), 128.50
(C,F)=23 Hz; CH), 124.95 (d, 3J
(s; CH), 128.72 (s; CH), 128.78 (s; CH), 135.70–138.71 (m; CF), 136.10
(s; CH), 138.74–141.78 (m; CF), 140.24 (d, J
149.13 (m; CF), 155.26 (brs; CB), 156.32 (s; CN), 164.90 ppm (d,
CB), 166.70 ppm (s; quaternary C); 19F NMR (376 MHz, CDCl3, CCl3F):
4
ACHTUNGERTN(NUNG C,F)=2.9 Hz; CN), 146.16–
3
d=À162.38 to À162.18 (m, 4F), À156.00 (t, J
N
to À131.07 ppm (m, 4F); UV/Vis (hexane): lmax (e)=265 (8.8ꢀ103),
443 nm (2.4ꢀ104 dm3 molÀ1 cmÀ1); MS (EI, 70 eV): m/z (%): 556 (100)
[M+], 541 (23), 525 (6), 513 (15), 464 (11), 389 (11), 321 (15), 241 (17), 77
(67), 51 (13); elemental analysis calcd (%) for C25H11BF10N2O: C 53.99,
H 1.99, N 5.04; found: C 54.17, H 2.21, N 5.05.
1J
ACHTUNGTRENNUNG
to À161.75 (m, 4F), À155.33 (t, 3J
(F,F)=21 Hz, 2F), À131.10 to À130.94
G
(m, 4F), À104.11 ppm (s, 1F); UV/Vis (hexane): lmax (e)=241 (7.3ꢀ103),
390 nm (1.1ꢀ104 dm3 molÀ1 cmÀ1); MS (EI, 70 eV): m/z (%): 544 (90)
[M+], 525 (11), 95 (100), 75 (18); HRMS (FAB+): m/z calcd for
C24H8BF11N2: 544.0605; found 544.0612; elemental analysis calcd (%) for
C24H8BF11N2: C 52.98, H 1.48, N 5.15; found: C 53.02, H 1.71, N 4.92.
Synthesis of [4-bromo-2-(phenylazo)phenyl]bis(pentafluorophenyl)bo-
AHCTUNGERTGrNNUN ane (1i): A similar reaction to that described for the preparation for 1d
by using 2i (500 mg, 1.29 mmol) in Et2O (80 mL) with nBuLi (1.6m in
hexane, 0.85 mL, 1.4 mmol) and 3b (0.35 mL, 1.4 mmol) gave 1i (284 mg,
36%). Brown solid; m.p. 193.8–195.38C; 1H NMR (400 MHz, CDCl3,
Synthesis of [2-(4-butylphenylazo)phenyl]bis(pentafluorophenyl)borane
(1 f): A similar reaction to that described for the preparation for 1d by
using 2 f (1.02 g, 2.80 mmol) in Et2O (40 mL) with nBuLi (1.6m in
hexane, 1.90 mL, 3.04 mmol) and 3b (0.820 mL, 3.11 mmol) in Et2O
(10 mL) gave 1 f (769 mg, 47%). Brown solid; m.p. 126.6–127.58C
TMS): d=7.47 (t, 3J
7.67 (d, 3J
2H), 8.17 ppm (d, 3J
BF3·OEt2): d=À0.9 ppm (line width
G
ACHTUNGTRENNUNG
E
ACHTUNGTRENNUNG
(decomp); 1H NMR (400 MHz, CDCl3, TMS): d=0.93 (t, 3J
A
AHCTUNGTRENNUNG
3
h
7.6 Hz, 3H), 1.37 (sext, J
G
R
3J
3J
3J
ACHTUNGTRENNUNG
(100 MHz, CDCl3, TMS): d=114.32 (brs; CB), 123.32 (s; CH), 130.22 (s;
CH), 130.49 (s; CH), 132.42 (s; CH), 132.86 (s; CH), 133.71 (s; CH),
133.87 (s; quaternary C), 136.63–139.13 (m; CF), 139.59–142.24 (m; CF),
144.36 (s; quaternary C), 147.06–149.46 (m; CF), 155.43 (s; quaternary
C), 157.72 ppm (brs; CB); 19F NMR (376 MHz, CDCl3, CCl3F): d=
A
U
ACHTUNGTRENNUNG
A
U
ACHTUNGTRENNUNG
7.92–7.96 (m, 2H), 8.29 ppm (d, 3J
U
(128 MHz, CDCl3, BF3·OEt2): d=À0.5 ppm (line width h1/2 =304 Hz);
13C{1H} NMR (126 MHz, CDCl3, TMS): d=13.88 (s; CH3), 22.42 (s;
CH2), 32.97 (s; CH2), 35.62 (s; CH2), 114.43 (brs; CB), 122.59 (s; CH),
128.32 (s; CH), 128.51 (s; CH), 129.71 (s; CH), 135.52 (s; CH), 135.81–
138.40 (m; CF), 138.65–141.37 (m; CF), 141.94 (s; quaternary C), 146.40–
148.82 (m; CF), 148.99 (s; quaternary C), 154.85 (brs; CB), 156.26 ppm
À163.53 (t, 3J
N
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
(7.6ꢀ103), 416 nm (2.3ꢀ104 dm3 molÀ1 cmÀ1); MS (EI, 70 eV): m/z (%):
606 (100) [M+], 604 (81); elemental analysis calcd (%) for
C24H8BBrF10N2: C 47.64, H 1.33, N 4.63; found: C 47.45, H 1.57, N 4.47.
Chem. Eur. J. 2010, 16, 5026 – 5035
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
5033

N. Kano, T. Kawashima, M. Asashima et al.
Synthesis of bis(pentafluorophenyl)[3-(phenylazo)biphen-4-yl]borane
(1j): A solution of borane 1i (79 mg, 0.13 mmol), phenylboronic acid
(64 mg, 0.52 mmol), and potassium carbonate (54 mg, 0.39 mmol) in tolu-
(1.2ꢀ104), 474 nm (5.8ꢀ103 dm3 molÀ1 cmÀ1); MS (FAB+): m/z: 542 [M+];
elemental analysis calcd (%) for C24H9BF10N2O: C 53.17, H 1.67, N 5.17;
found: C 53.20, H 1.86, N 5.19.
ene (5 mL) was stirred for 15 min at room temperature. Tetrakis(tri
ACHTUNGTRENNUNG
ACHTUNGTNERpNUNG hen-
X-ray crystallographic analysis: X-ray diffraction data for single crystals
of 1a,[11] 1b, and 1d were collected using Rigaku MERCURY CCD. The
structures were solved by the direct method and refined by full-matrix
least-squares using SHELX-97.[21]
and the solution was stirred for 8 h at 908C, and then heated to refluxed
for 14 h. After the reaction was quenched by aqueous ammonium chlo-
ride, the reaction mixture was extracted with Et2O. The organic layer was
dried over anhydrous magnesium sulfate and the solvent was evaporated.
Purification of the residue with silica gel chromatography (eluent:
¯
Crystal data for 1a: C24H9BF10N2, Mr =526.14, triclinic, space group P1,
a=9.861(7), b=10.732(6), c=10.822(7) ꢂ, a=99.976(7), b=113.714(8),
g=91.109(7)8, V=1027.7(11) ꢂ3, Z=2, 1calcd =1.700 gcmÀ3
,
m=
1
hexane) gave 1j (63 mg, 80%). Orange solid; m.p. 82.1–83.88C; H NMR
0.164 mmÀ1, T=120(2) K, 6375 reflections measured, 3488 unique, final
R1 [I>2s(I)]=0.0371, wR2 (all data)=0.1068, GOF=1.089.[22]
(400 MHz, CDCl3, TMS): d=7.39–7.53 (m, 6H), 7.63 (d, 3J
ACHTUNGTRENNUNG
8.0 Hz, 2H), 7.76 (d, 3J
3J
ACHTUNGTRENNUNG
Crystal data for 1b: C25H11BF10N2O, Mr =556.17, monoclinic, space group
P21/c, a=9.7613(9), b=15.8245(13), c=14.7521(14) ꢂ, b=103.6037(13)8,
V=2214.8(3) ꢂ3, Z=4, 1calcd =1.668 gcmÀ3, m=0.160 mmÀ1, T=120(2) K,
24521 reflections measured, 5071 unique, final R1 [I>2s(I)]=0.0436,
wR2 (all data)=0.1031, GOF=1.092.[22]
N
ACHTUNGTRENNUNG
(128 MHz, CDCl3, BF3·OEt2): d=À0.7 ppm (line width h1/2 =142 Hz);
13C{1H} NMR (126 MHz, CDCl3, TMS): d=115.20 (brs; CB), 123.19 (s;
CH), 127.65 (s; CH), 128.47 (s; CH), 129.71 (s; CH), 129.74 (s; CH),
129.87 (s; CH), 130.43 (s; CH), 133.15 (s; CH), 136.90–138.88 (m; CF),
139.83–141.82 (m; CF), 140.36 (s; quaternary C), 144.58 (s; quaternary
C), 147.44–149.42 (m; CF), 149.43 (s; quaternary C), 156.55 (s; quaterna-
ry C), 157.65 ppm (brs; CB); one signal of the tertiary carbon atom in ar-
omatic region was not observed due to overlapping; 19F NMR (376 MHz,
Crystal data for 1d: C25H8BF13N2, Mr =594.14, monoclinic, space group
P21/c, a=13.015(5), b=11.864(5), c=15.083(7) ꢂ, b=97.4536(19)8, V=
2309.3(17) ꢂ3, Z=4, 1calcd =1.709 gcmÀ3 m=0.175 mmÀ1
, , T=120(2) K,
17629 reflections measured, 5184 unique, final R1 [I>2s(I)]=0.0446,
wR2 (all data)=0.0901, GOF=0.844.[22]
CDCl3, CCl3F): d=À163.88 (dt, 3J
(F,F)=22 Hz, 4J
ACHUTGTNRENNUG CAHTUNGTRENNUNG
À157.55 (t, 3J
(F,F)=22 Hz, 2F), À132.16 ppm (dt, 3J
ACHTUNGTRENNUNG
Theoretical calculations: All calculations were carried out at DFT level
using the B3PW91[23,24] exchange-correlation functional implemented in
the Gaussian 03 (Revision C.02)[25] suite of programs. Geometry optimi-
zations were performed with the 6-31G(d) basis set and other all calcula-
tions based on the optimized structures were performed with the 6-31+
G(d) basis set. The energy levels of excited states were obtained by a
time-dependent DFT (TD-DFT) method. Zero-point energy corrections
are not included in all the calculations.
ACHTUNGTRENNUNG
4J(F,F)=7.5 Hz, 4F); UV/Vis (hexane) lmax (e)=251 (7.5ꢀ103), 441 nm
(2.2ꢀ104 dm3 molÀ1 cmÀ1); MS (FAB+): m/z: 602 [M+]; elemental analysis
calcd (%) for C30H13BF10N2: C 59.83, H 2.18, N 4.65; found: C 59.47, H
2.16, N 4.27.
Synthesis of [4-(diphenylamino)-2-(phenylazo)phenyl]bis(pentafluoro-
phenyl)borane (1k): A mixture of 1i (150 mg, 0.248 mmol), diphenyl-
ACHTUNGTRENNUNG
Xenopus embryo manipulation: In vitro fertilization, embryo culture, and
microinjection were carried out as described previously.[26] Azobenzene
1b (10 mg) was dissolved in DMSO or ethanol (1 mL) for the injection
solution; these azobenzene solutions were diluted tenfold by using dis-
tilled water. An aqueous solution of GFP mRNA (0.04 mgmLÀ1) was
prepared by using distilled water as control. These three solutions were
injected (5 nL) into a blastomere. Animal experiments in this study are
approved in AIST (accreditation number: 2009-109B).
ACHTUNGTRENNUNG
1058C for 7 h. The usual workup followed by purification by silica gel
chromatography (eluent: hexane) gave 1k (100 mg, 58%). Purple solid;
m.p. 134.3–137.58C; H NMR (400 MHz, CDCl3, TMS): d=7.06–7.07 (m,
1
1H), 7.14–7.16 (m, 4H), 7.22–7.27 (m, 3H), 7.30–7.37 (m, 7H), 7.80 (d,
3J(H,H)=8.4 Hz, 2H), 7.95 ppm (d, 3J(H,H)=8.8 Hz, 1H); 11B NMR
ACHTUNGTRENNUNG ACHTUNGTRENNUGN
(128 MHz, CDCl3, BF3·OEt2): d=À3.2 ppm (line width h1/2 =260 Hz);
13C{1H} NMR (100 MHz, CDCl3, TMS): d=116.82 (s; CH), 118.42 (s;
CH), 121.06 (s; CH), 126.67 (s; CH), 126.91 (s; CH), 129.24 (s; CH),
129.56 (s; CH), 129.78 (s; CH), 130.65 (s; CH), 135.80–138.35 (m; CF),
138.35–140.97 (m; CF), 143.99 (s; quaternary C), 144.70 (s; quaternary
C), 146.44–148.83 (m; CF), 150.71 (s; quaternary C), 154.94 ppm (s; qua-
ternary C); two signals of the carbon adjacent to the boron atom were
Acknowledgements
not observed due to broadening; 19F NMR (376 MHz, CDCl3, CCl3F):
3
d=À162.32 to À162.19 (m, 4F), À156.98 (t, J
(F,F)=22 Hz, 2F), À131.16
We thank Dr. Tomohisa Takaya, The University of Tokyo, for his help in
measurement of the fluorescence lifetimes. We thank Tosoh Finechem
Corp. and Central Glass Co., Ltd. for gifts of alkyllithiums and fluorine
compounds, respectively. This work was partially supported by Grants-in-
Aid for the Global COE Program for Chemistry Innovation and for Sci-
entific Researches from the Ministry of Education, Culture, Sports, Sci-
ence and Technology, Japan, the Japan Society for the Promotion of Sci-
ence, and JGC-S Scholarship Foundation. J.Y. was granted a Research
Fellowship of Japan Society for the Promotion of Science for Young Sci-
entists.
to À131.07 ppm (m, 4F); UV/Vis (hexane) lmax (e)=281 (1.5ꢀ104),
553 nm (2.8ꢀ104 dm3 molÀ1 cmÀ1); MS (FAB+): m/z: 639 [M+]; elemental
analysis calcd (%) for: C 62.36, H 2.62, N 6.06; found: C 62.24, H 2.90, N
5.96.
Synthesis of bis(pentafluorophenyl)[2-(phenylazo)phenoxy]borane (5): A
solution of 4 (198 mg, 1.00 mmol) and 3b (0.264 mL, 1.00 mmol) in tolu-
ene (30 mL) was heated to reflux for 14 h in a flask equipped with a
Soxhlet extractor containing molecular sieves 4 ꢂ. Evaporation of the
solvent and recrystallization from Et2O afforded 5 (361 mg, 67%). Red
crystals (Et2O); m.p. 148.9–149.78C (decomp); 1H NMR (400 MHz,
CDCl3, TMS): d=6.98 (dd, 3J
(ddd, 3J(H,H)=8.1 Hz, 3J(H,H)=7.1 Hz, 4J
7.40 (m, 2H), 7.42–7.47 (m, 1H), 7.55–7.61 (m, 3H), 7.88 ppm (dd,
3J(H,H)=8.1 Hz, 4J(H,H)=1.7 Hz, 1H); 11B NMR (128 MHz, CDCl3,
BF3·OEt2): d=0.6 ppm (line width
1/2 =112 Hz); 13C{1H} NMR
G
ACHTUNGERTN(NUNG H,H)=1.0 Hz, 1H), 7.09
E
R
ACHTUNGTRENNUNG
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5034
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ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 5026 – 5035

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Received: January 29, 2010
Published online: April 14, 2010
Chem. Eur. J. 2010, 16, 5026 – 5035
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
5035