Environment-Sensitive Fluorescent Probe: A Benzophosphole Oxide with an Electron-Donating Substituent

Article abstract of DOI:10.1002/anie.201500229

Electron-donating aryl groups were attached to electron-accepting benzophosphole skeletons. Among several derivatives thus prepared, one benzophosphole oxide was particularly interesting, as it retained high fluorescence quantum yields even in polar and p

Full text of DOI:10.1002/anie.201500229

Angewandte
Chemie
DOI: 10.1002/anie.201500229
Phosphorus Heterocycles
Environment-Sensitive Fluorescent Probe: A Benzophosphole Oxide
with an Electron-Donating Substituent**
Eriko Yamaguchi, Chenguang Wang, Aiko Fukazawa,* Masayasu Taki, Yoshikatsu Sato,
Taeko Sasaki, Minako Ueda, Narie Sasaki, Tetsuya Higashiyama,* and Shigehiro Yamaguchi*
Abstract: Electron-donating aryl groups were attached to
electron-accepting benzophosphole skeletons. Among several
derivatives thus prepared, one benzophosphole oxide was
particularly interesting, as it retained high fluorescence quan-
tum yields even in polar and protic solvents. This phosphole-
based compound exhibited a drastic color change of its
fluorescence spectrum as a function of the solvent polarity,
while the absorption spectra remained virtually unchanged.
Capitalizing on these features, this phosphole-based compound
was used to stain adipocytes, in which the polarity of
subcellular compartments could then be discriminated on the
basis of the color change of the fluorescence emission.
phosphine oxide and sulfide derivatives exhibit desirably high
thermal and chemical stability. Taking advantage of these
features, phosphole-based materials have been widely applied
[
1g,h]
in organic electronics,
including organic light-emitting
[3,4]
[5]
diodes
and photovoltaics. However, biological applica-
tions have not yet been explored exhaustively. In this context,
we would like to disclose here the successful development of
highly fluorescent phosphole derivatives, and demonstrate
their potential as fluorescent bioimaging probes.
The combination of an electron-accepting p-skeleton with
an electron-donating moiety represents a rational design
approach for producing environment-sensitive fluorescent
[6]
molecules. Following this avenue, we have now designed the
P
hosphole, a heavier phosphorus analogue of pyrrole, is an
attractive building block for p-conjugated materials.
[1]
Whereas pyrrole is aromatic and has electron-donating
properties, phosphole is nonaromatic and exhibits an elec-
[
1b,2]
tron-accepting character.
Relative to pyrrole, the lone-
pair electrons of the pyramidalized phosphorus atom in the
phosphole conjugate to a lesser extent with the p-system.
Instead, effective orbital interaction occurs between the
p* orbital of a butadiene moiety and the s* orbital of an
exocyclic s bond on the phosphorus atom, thus resulting in
a decreased LUMO level. The electron-accepting properties
of phosphole can be further increased by facile transforma-
tions into phosphine oxides, phosphine sulfides, phosphonium
salts, and metal complexes. Among these, especially the
[
*] E. Yamaguchi, Prof. Dr. A. Fukazawa, Prof. Dr. S. Yamaguchi
Department of Chemistry, Graduate School of Science
Nagoya University
Furo, Chikusa, Nagoya 464-8602 (Japan)
E-mail: aiko@chem.nagoya-u.ac.jp
yamaguchi@chem.naogya-u.ac.jp
T. Sasaki, Prof. Dr. N. Sasaki, Prof. Dr. T. Higashiyama
Division of Biological Science, Graduate School of Science
Nagoya University, Furo, Chikusa, Nagoya 464-8602 (Japan)
C. Wang, Prof. Dr. M. Taki, Dr. Y. Sato, Dr. M. Ueda,
Prof. Dr. T. Higashiyama, Prof. Dr. S. Yamaguchi
Institute of Transformative Bio-Molecules (WPI-ITbM)
Nagoya University, Furo, Chikusa, Nagoya 464-8602 (Japan)
E-mail: higashi@bio.nagoya-u.ac.jp
[
**] This work was supported by JST, CREST (S.Y.), a Grant-in-Aid for
Scientific Research on Innovative Areas “Organic Synthesis based
on Reaction Integration” (no. 2105) from MEXT (Japan) (A.F.), and
the Japan Advanced Plant Science Network. E.Y. thanks the JSPS for
a Research Fellowship for Young Scientists. ITbM is supported by
the World Premier International Research Center (WPI) Initiative,
Japan.
Figure 1. a) Chemical structures of the benzophosphole derivatives 1–
4
and benzothiophene dioxide congener 5. b) Comparison of represen-
tative environment-sensitive fluorescent probes, whereby circles and
squares represent the maxima of absorption and emission, respec-
tively. Empty and filled symbols denote wavelengths in nonpolar and
polar solvents, respectively. [a] Reference [8b]; [b] Reference [9b,c];
[c] Reference [10]; [d] Reference [11b].
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201500229.
Angew. Chem. Int. Ed. 2015, 54, 1 – 6
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1
These are not the final page numbers!

.
Angewandte
Communications
environment-sensitive fluorescent molecule 1 by combining
an electron-accepting benzophosphole oxide with an elec-
tron-donating triphenylamine substituent (Figure 1a). Envi-
ronment-sensitive fluorescent probes are particularly impor-
tant for the visualization of polarity changes associated with
position, in moderate to good yields. An ensuing Suzuki–
Miyaura cross-coupling reaction of 7 with phenylboronic acid
under standard reaction conditions afforded the derivatives
1 and 2 in good yields. The compound 1 was further
transformed into the corresponding phosphole sulfide 3 by
treatment with Lawessonꢀs reagent. An analogous phospho-
nium derivative 4 was also prepared by the reduction of 1 with
[
7]
cellular events, and the general requirements for such
probes are: 1) high fluorescence quantum yields (F ) irre-
F
spective of the solvent polarity, 2) large Stokes shifts to avoid
autofluorescence interference, and 3) drastic color change of
the fluorescence emission as a function of the solvent polarity.
HSiCl , and subsequent treatment with methyl triflate. The
3
structures of these compounds were assigned unequivocally
based on the results from NMR spectroscopy and mass
spectrometry measurements.
The p-(diphenylamino)phenyl-substituted benzophos-
phole oxide 1 exhibited excellent fluorescent properties
(Table 1). In toluene, the absorption maximum of 1 was
[8]
Several previously reported fluorophores, such as prodan,
[
9]
[10]
[11]
1
,8-ANS, dapoxyl, and Nile red, are known to satisfy
these requisites. However, in comparison to these, 1 offers the
following additional advantages: 1) compatibility with
4
05 nm light-emitting diode lasers, which are frequently
used for excitation in fluorescence microscopy, and 2) bath-
ochromically shifted reddish fluorescence emission as a result
of excitation with such a 405 nm laser (Figure 1b). Herein, we
discuss the key structural requisites necessary for our new
fluorophore to exhibit its characteristic fluorescence proper-
ties, and we also demonstrate its application in cell imaging.
An important advantage of benzophosphole oxide as an
electron-accepting building block is the facile synthetic access
to its skeleton. Recently, several new reactions have been
Table 1: Photophysical properties of 1–5 in various solvents.
Cmpd Solvents labs
e
lem
FF[b] eꢀF
F
[
a]
4
À1
À1
4
À1
À1
[nm]
[10 m cm
]
[nm]
[10 m cm
]
1
toluene 415
1.87
1.74
1.73
1.73
1.60
1.66
1.03
0.94
1.58
1.28
1.34
1.23
2.56
2.29
528 0.94 1.8
553 0.94 1.6
565 0.90 1.6
575 0.84 1.5
593 0.58 0.9
601 0.64 1.1
471 0.74 0.8
490 0.69 0.7
526 0.87 1.4
608 0.61 0.8
672 0.20 0.3
702 0.02 0.02
507 0.92 2.4
628 0.06 0.1
CHCl3
420
415
CH Cl
2
2
acetone 403
ethanol 417
DMSO
toluene 364
DMSO 364
toluene 412
DMSO 411
toluene 448
DMSO 440
toluene 419
DMSO 418
412
[
12]
developed for the synthesis of benzophospholes.
Our
2
3
4
5
contributions to this area include a versatile synthesis
for benzophospholes based on the intramolecular trans-
[13,14]
halophosphanylation of o-phosphanylphenylacetylenes,
which afforded a series of donor–acceptor-type benzophos-
pholes. Thus, starting from the (o-bromophenyl)(p-substi-
tuted phenyl)acetylene precursors 6, a lithiation with two
equivalents of tBuLi, followed by treatment with PhP-
[
a] Longest wavelength absorption maximum. [b] Absolute fluorescence
quantum yields determined with a calibrated integrating sphere system
errors <3%).
(
NEt )Cl generated the corresponding o-aminophosphanyl-
2
substituted intermediates (Scheme 1). Without isolation,
(
a subsequent treatment with PBr resulted in the halogen-
3
ation of the phosphorus moiety, followed by an intramolec-
ular cyclization by trans-halophosphanylation. This conven-
ient one-pot procedure furnished the 3-bromobenzo[b]phos-
phole oxides 7, bearing electron-donating aryl groups at the 2-
observed at labs = 415 nm, while an intense fluorescence band
with a fluorescence quantum yield (F ) of 0.94 was recorded
F
at lem = 528 nm. With increasing solvent polarity, the absorp-
tion spectra showed only subtle changes (labs = 403–420 nm),
whereas the fluorescence bands were shifted significantly in
a bathochromic direction (DMSO: 601 nm; EtOH: 593 nm).
Most notably, 1 preserves high fluorescence quantum yields in
polar (DMSO: F = 0.64) and protic solvents (EtOH: F =
F
F
0
7
.58) despite the corresponding large Stokes shifts (DMSO:
630 cm ; EtOH: 7120 cm ). The fact that a brightness of
À1
À1
the fluorescence, denoted by the product of molar extinction
[
15]
4
coefficient (e) and FF, of about 10 was attained even in
ethanol, thus suggests potential applications for this com-
pound as a fluorescent probe.
A comparison of 1 with related compounds, including the
anisyl-substituted 2, containing a weaker electron-donating
substituent, as well as with the electron-accepting benzo-
phosphole sulfide 3, phosphonium salt 4, and thiophene
dioxide congener 5, demonstrates that the well-balanced
combination of the electron-accepting phosphole oxide and
the electron-donating diphenylamino group in 1 is, in more
than one way, crucial for the presence of the observed
outstanding photophysical properties (Table 1; for exhaustive
Scheme 1. Synthesis of the donor-substituted benzophosphole deriva-
tives 1–4. Tf=trifluoromethanesulfonyl.
2
www.angewandte.org
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2015, 54, 1 – 6
These are not the final page numbers!

Angewandte
Chemie
data, see Table S1 in the Supporting Information). Firstly,
a comparison between 1 and 2 shows the importance of the
electron-donating properties of the diphenylamino group in 1.
While the absorption spectra of the anisyl derivative 2 showed
subtle solvatochromism (labs = 359–367 nm), the emission
spectra showed a bathochromic shift with increasing solvent
for the radiative (k ) and nonradiative (k ) decay were
r nr
determined based on fluorescence quantum yield and lifetime
values (t). While the k values in DMSO are comparable for 1,
r
8
À1
4, and 5 (0.89–1.1 ꢁ 10 s ), the k values of 4 and 5 (52 ꢁ
nr
8
À1
8
À1
10 s and 15 ꢁ 10 s , respectively) are much higher than
8
À1
that of 1 (0.60 ꢁ 10 s ). These results indicate that even
though the presence of a strong electron-accepting ring
skeleton is crucial for the bathochromically shifted emission,
excessive electron-accepting properties may accelerate the
nonradiative decay process from the excited singlet state.
Although the photophysical properties of 1 and 3 are
almost comparable, their solubility in polar solvents differs
greatly. The solubility of phosphole oxide 1 in DMSO
polarity. However, the shift for 2 from toluene to ethanol
À1
(
1310 cm ) was measured to be significantly smaller than
À1
that in 1 (2080 cm ). Based on TD-DFT calculations at the
PBE0/6-31 + G(d) level of theory, the HOMO of 1 is mostly
localized on the electron-donating triphenylamine moiety,
whereas that of 2 is delocalized over the 2-anisylbenzophosp-
hole moiety. Accordingly, the character of S –S transitions in
0
1
À1
1
exhibits the intramolecular charge transfer (ICT) character
(17.4 gL ) is significantly higher than that of phosphole
sulfide 3 (0.8 gL ). This difference most probably results
À1
from the triphenylamine moiety to the electron-accepting
benzophosphole oxide moiety, whereas that of 2 is attribut-
able to p–p* transition. The calculated values for the
oscillator strengths are 0.396 and 0.265 in 1 and 2, respectively.
The higher value for 1 is consistent with a larger molar
from the more polarized P=O moiety relative to the P=S
moiety. Electron densities on the P, O, and S atoms in 1 and 3
[16]
were calculated by natural population analysis, using the
B3LYP hybrid functional in combination with the 6-311G(d)
basis set. For the P and O atoms in 1, electron density values
of 1.88 and À1.06, respectively, were calculated, whereas in 3,
values of 1.32 and À0.58 were obtained for the P and S atoms,
respectively.
4
À1
À1
extinction coefficient for 1 (e = 1.87 ꢁ 10 m cm ) compared
4
À1
À1
to 2 (e = 1.03 ꢁ 10 m cm ), which should, at least in part,
account for the higher quantum yield of 1 relative to 2.
Secondly, a comparison of 1 with the phosphole sulfide 3
and phosphonium salt 4 illustrates the impact of the electron-
accepting character of the benzophosphole moiety. In terms
of the LUMO levels, the parent phosphole rings with P=O or
P=S moieties should possess a comparable electron-accepting
An advantageous feature of 1 for the application as
a fluorescent bioimaging probe is the drastic color change of
the fluorescence emission as a function of the solvent polarity,
that is, the color change from a bluish green in toluene to
a reddish orange in DMSO (Figure 2a). Notably, the Lippert–
character (see Figure S10 in the Supporting Information).
Commensurate with this similarity, 1 and 3 exhibit compara-
ble photophysical properties. For example, 3 shows high
[6]
Mataga plot for 1 in various solvents, including the protic
2
solvents ethanol and methanol, showed high linearity (R =
fluorescence quantum yields both in polar (DMSO: F =
0.93; Figure 2b). This linearity suggests that the specific
F
0
.61) and in protic (EtOH: F = 0.76) solvents, and the
F
maximum fluorescence emission wavelengths are comparable
to those of 1. In contrast, 4 exhibits, even in toluene, a 33 nm
bathochromically shifted absorption maximum, as well as
a 144 nm red-shifted fluorescence maximum relative to that
of 1 (see Figure S4 in the Supporting Information and
Table 1). These large differences are obviously due to the
much stronger electron-accepting character of the phospho-
nium moiety. Irrespective of the solvent polarity, 4 exhibits
substantially lower F values associated with these red-
F
shifted emissions.
Thirdly, a comparison with the thiophene dioxide con-
gener 5 provides insight into the crucial role of the P=O
moiety with respect to the strong fluorescence. The parent
thiophene dioxide has a lower LUMO level compared to that
of phosphole oxide (Figure S10). Whereas 5 exhibits, in
nonpolar solvents, a maximum emission wavelength at
slightly shorter wavelengths compared to 1, its maximum
emission wavelength in DMSO is bathochromically shifted by
3
5 nm (see Figure S5 in the Supporting Information). An even
more significant difference is observed for the fluorescence
quantum yield of 5 in polar or protic solvents, which is much
lower than that of 1 (Table 1).
These comparisons clearly demonstrate that the use of
a sufficiently strong electron-accepting ring is crucial to
preserving intense fluorescence in polar or protic solvents.
The fluorescence intensity in such solvents should largely rely
on the dynamics of the excited state. Therefore, rate constants
Figure 2. Solvent dependence of the fluorescence of 1: a) Photos of
the color change of the fluorescence emission of 1 in various solvents.
b) A Lippert–Mataga plot for 1 in various solvents (R =0.93), whereby
2
Dn and Df represent the Stokes shift (nabsÀn ) and the orientation
em
2
2
polarizability of solvents (eÀ1)/(2e+1)À(n À1)/(2n +1), respectively.
e=dielectric constant, n=refractive index.
Angew. Chem. Int. Ed. 2015, 54, 1 – 6
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
3
These are not the final page numbers!

.
Angewandte
Communications
solvent effect, resulting from the hydrogen bonding in protic
change in subcellular compartments during the adipogenic
solvents, is not significant despite the presence of the polar P= differentiation process. Incubation of 3T3-L1 cells with 1 mm
O moiety. Therefore, a simple monitoring of the emission
maxima should allow discrimination of polarity changes/
differences in biological media.
To evaluate the utility of 1 as a fluorescent probe for the
environmental polarity in biological systems, the dependence
of the fluorescence spectra of 1 on the pH value was
examined in aqueous solutions. Because of the low solubility
of 1 in water, a mixed solvent system, consisting of DMSO
and pH-buffered solution (7:3 v/v), was used. The emission
spectrum remained unchanged for a wide range of pH values
of 1 in Dulbeccoꢀs modified Eagleꢀs medium (DMEM) for
2 hours at 378C revealed a diffuse staining pattern with
several bright spots (0 day; Figure 3a), thus suggesting that
1 should be membrane permeable. The fluorescence spectra
of the bright spots (e.g. the spot indicated by the green circle)
exhibited an emission maximum between l = 521 and 530 nm,
whereas the emission spectrum in other intracellular domains
(e.g. indicated by the red circle) exhibited emission maxima at
a longer wavelength (l = 556–565 nm). Using these spectral
properties, the image was unmixed into two components
shown in green (Figure 3b) and orange (Figure 3c), respec-
tively. The difference in the emission properties clearly
demonstrates that 1 allows a discrimination of the environ-
mental polarity in the cells, as the polarity of the former
compartment is lower than that of the latter. As the differ-
entiation of 3T3-L1 cells into adipocytes proceeded, lipid
droplets increased in both number and size were found in the
bright-field images (Figure 3d). The deconvoluted images,
using the selected pixels with green color (Figure 3b),
strongly support that probe 1 is dissolved in the hydrophobic
lipid droplets and thus signals their polarity. In contrast, the
emission intensities in the other domains gradually decreased
during the adipogenic differentiation (Figure 3c), which may
correspond to a concentration decrease of filamentous actin
(
see Figure S6 in the Supporting Information), thus indicating
that the fluorescence properties of 1 are not affected by an
intracellular pH change. Subsequently, the photostability of
1
was investigated in ethanol under irradiation with a Xe
discharge lamp (300 W), and compared to the performance of
other representative dyes, such as BODIPY 493/503, fluo-
rescein, and prodan under the same experimental conditions.
The compound 1 was found to be more stable relative to all
the reference dyes (see Figure S7 in the Supporting Informa-
tion). Specifically, 1 showed an absorbance decrease of only
about 15% after 30 minutes of irradiation, whereas for
example, prodan was subjected to a decrease of more than
6
0%. Finally, an MTT assay showed that 1 exhibits no
cytotoxicity towards HeLa cells at concentrations below 5 mm,
which is high enough for cellular staining experiments (see
Figure S8 in the Supporting Information).
To demonstrate a practical application of 1, fluorescence
imaging of 3T3-L1 preadipocytes was examined using a con-
focal microscope equipped with a GaAsP multichannel
spectral detector (Figure 3). We expected to be able to
visualize the formation of fat in the cells, as the color change
of the fluorescence emission should correspond to the polarity
[17]
(F-actin). The observed sensitivity of 1 towards environ-
mental polarity, combined with suitable spectral imaging
techniques, should allow an elucidation of the molecular
mechanisms that control the balance between adipogenesis
[18]
and osteogenesis in mesenchymal stem cells.
In summary, a polarity-sensitive benzophosphole oxide-
based fluorophore with a high fluorescence quantum yield
was developed. While this benzophosphole oxide with an
electron-donating (diphenylamino)phenyl group shows an
almost constant absorption around labs = 405 nm, it also
exhibits a significant color change of the fluorescence
emission as a function of the polarity of the surrounding
environment. In addition, the benzophosphole oxide shows
a high photostability and no cytotoxicity. Moreover, we
demonstrated that this fluorescent probe allows discrimina-
tion of the cellular environment in adipocytes based on the
fluorescence emission color, which constitutes the first report
on staining living animal cells with phosphole-based fluores-
cence dyes. Future work will be directed towards the
enhancement of the water-solubility of these compounds, as
well as towards more bathochromically shifted emission
colors from further modifications at 3-position and/or from
using other benzophosphole skeletons.
Received: January 10, 2015
Published online: && &&, &&&&
Figure 3. Confocal microphotographs of 3T3L1 preadipocytes (top)
and adipocytes after 3 days (middle) and 8 days (bottom) of differ-
entiation. The cells were stained with 1 for 2 h prior to examination:
a) Lambda stack images in the emission range from l=416 to 689 nm
with excitation at 405 nm. b, c) Linear-deconvolution images using
reference spectra of selected pixels. The exhibited emission maxima in
the range of l=521–530 nm (green circle) and l=556–565 nm (red
circle), are shown in green and orange, respectively. d) Bright-field
images.
Keywords: fluorescent probes · imaging agents ·
.
phosphorus heterocycles · phosphole · solvatochromism
[1] For recent reviews, see: a) F. Mathey, Angew. Chem. Int. Ed.
2003, 42, 1578 – 1604; Angew. Chem. 2003, 115, 1616 – 1643;
4
www.angewandte.org
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2015, 54, 1 – 6
These are not the final page numbers!

Angewandte
Chemie
b) M. Hissler, P. W. Dyer, R. Rꢂau, Coord. Chem. Rev. 2003, 244,
[6] J. R. Lakowicz, Principles of Fluorescence Spectroscopy, 3rd ed.,
1
4
– 44; c) T. Baumgartner, R. Rꢂau, Chem. Rev. 2006, 106, 4681 –
727; d) J. Crassous, R. Rꢂau, Dalton Trans. 2008, 6865 – 6876;
Springer, 2006.
[7] a) L. A. Bagatolli, Biochim. Biophys. Acta Biomembr. 2006,
1758, 1541 – 1556; b) A. P. Demchenko, Y. Mꢂly, G. Duportail,
A. S. Klymchenko, Biophys. J. 2009, 96, 3461 – 3470; c) A. S.
Klymchenko, R. Kreder, Chem. Biol. 2014, 21, 97 – 113; d) G. S.
Loving, M. Sainlos, B. Imperiali, Trends Biotechnol. 2010, 28,
73 – 83.
e) Y. Matano, H. Imahori, Org. Biomol. Chem. 2009, 7, 1258 –
1
7
271; f) Y. Ren, T. Baumgartner, Dalton Trans. 2012, 41, 7792 –
800; g) T. Baumgartner, Acc. Chem. Res. 2014, 47, 1613 – 1622;
h) M. Stolar, T. Baumgartner, Chem. Asian J. 2014, 9, 1212 –
225.
1
[
[
2] L. Nyulꢃszi, Chem. Rev. 2001, 101, 1229 – 1246.
[8] a) G. Weber, F. J. Farris, Biochemistry 1979, 18, 3075 – 3078; b) Y.
Niko, S. Kawauchi, G. Konishi, Chem. Eur. J. 2013, 19, 9760 –
9765.
[9] a) G. Weber, D. J. R. Laurence, Biochem. J. 1954, 56, xxxi;
b) D. C. Turner, L. Brand, Biochemistry 1968, 7, 3381 – 3390;
c) S. H. Hꢄttenhain, W. Balzer, R. Feldmann, Z. Naturforsch. A
1994, 49, 1087 – 1090.
[10] Z. Diwu, Y. X. Lu, C. L. Zhang, D. H. Klaubert, R. P. Haugland,
Photochem. Photobiol. 1997, 66, 424 – 431.
[11] a) P. Greenspan, E. P. Mayer, S. D. Fowler, J. Cell Biol. 1985, 100,
965 – 973; b) J. Jose, K. Burgess, J. Org. Chem. 2006, 71, 7835 –
7839.
3] a) C. Fave, T. Y. Cho, M. Hissler, C. W. Chen, T. Y. Luh, C. C.
Wu, R. Rꢂau, J. Am. Chem. Soc. 2003, 125, 9254 – 9255; b) H. C.
Su, O. Fadhel, C. J. Yang, T. Y. Cho, C. Fave, M. Hissler, C. C.
Wu, R. Rꢂau, J. Am. Chem. Soc. 2006, 128, 983 – 995; c) O.
Fadhel, M. Gras, N. Lemaitre, V. Deborde, M. Hissler, B.
Geffroy, R. Rꢂau, Adv. Mater. 2009, 21, 1261 – 1265; d) D. Joly,
D. Tondelier, V. Deborde, B. Geffroy, M. Hissler, R. Rꢂau, New
J. Chem. 2010, 34, 1603 – 1611; e) D. Joly, D. Tondelier, V.
Deborde, W. Delaunay, A. Thomas, K. Bhanuprakash, B.
Geffroy, M. Hissler, R. Rꢂau, Adv. Funct. Mater. 2012, 22,
567 – 576; f) H. Chen, W. Delaunay, J. Li, Z. Y. Wang, P. A.
Bouit, D. Tondelier, B. Geffroy, F. Mathey, Z. Duan, R. Rꢂau, M.
Hissler, Org. Lett. 2013, 15, 330 – 333; g) H. Tsuji, K. Sato, Y.
Sato, E. Nakamura, J. Mater. Chem. 2009, 19, 3364 – 3366.
[12] a) T. Sanji, K. Shiraishi, T. Kashiwabara, M. Tanaka, Org. Lett.
2008, 10, 2689 – 2692; b) H. Tsuji, K. Sato, L. Ilies, Y. Itoh, Y.
Sato, E. Nakamura, Org. Lett. 2008, 10, 2263 – 2265; c) Y. R.
Chen, W. L. Duan, J. Am. Chem. Soc. 2013, 135, 16754 – 16757;
d) Y. Unoh, K. Hirano, T. Satoh, M. Miura, Angew. Chem. Int.
Ed. 2013, 52, 12975 – 12979; Angew. Chem. 2013, 125, 13213 –
13217; e) B. Wu, M. Santra, N. Yoshikai, Angew. Chem. Int. Ed.
2014, 53, 7543 – 7546; Angew. Chem. 2014, 126, 7673 – 7676.
[13] A. Fukazawa, Y. Ichihashi, Y. Kosaka, S. Yamaguchi, Chem.
Asian J. 2009, 4, 1729 – 1740.
[
4] a) Y. Matano, T. Miyajima, T. Fukushima, H. Kaji, Y. Kimura, H.
Imahori, Chem. Eur. J. 2008, 14, 8102 – 8115; b) A. Saito, T.
Miyajima, M. Nakashima, T. Fukushima, H. Kaji, Y. Matano, H.
Imahori, Chem. Eur. J. 2009, 15, 10000 – 10004; c) Y. Matano, A.
Saito, T. Fukushima, Y. Tokudome, F. Suzuki, D. Sakamaki, H.
Kaji, A. Ito, K. Tanaka, H. Imahori, Angew. Chem. Int. Ed. 2011,
50, 8016 – 8020; Angew. Chem. 2011, 123, 8166 – 8170.
[
5] a) A. Kira, Y. Shibano, S. Kang, H. Hayashi, T. Umeyama, Y.
Matano, H. Imahori, Chem. Lett. 2010, 39, 448 – 450; b) Y.
Matano, A. Saito, Y. Suzuki, T. Miyajima, S. Akiyama, S. Otsubo,
E. Nakamoto, S. Aramaki, H. Imahori, Chem. Asian J. 2012, 7,
[14] T. Butters, W. Winter, Chem. Ber. 1984, 117, 990 – 1002.
[15] L. D. Lavis, R. T. Raines, ACS Chem. Biol. 2008, 3, 142 – 155.
[16] A. E. Reed, R. B. Weinstock, F. Weinhold, J. Chem. Phys. 1985,
83, 735 – 746.
2
305 – 2312; c) Y. Matano, H. Ohkubo, Y. Honsho, A. Saito, S.
[17] H. Sonowal, A. Kumar, J. Bhattacharyya, P. K. Gogoi, B. G.
Jaganathan, J. Biomed. Sci. 2013, 20, 71 – 80.
[18] M. Kawai, C. J. Rosen, Nat. Rev. Endocrinol. 2010, 6, 629 – 636.
Seki, H. Imahori, Org. Lett. 2013, 15, 932 – 935; d) Y. Matano, H.
Ohkubo, T. Miyata, Y. Watanabe, Y. Hayashi, T. Umeyama, H.
Imahori, Eur. J. Inorg. Chem. 2014, 1620 – 1624; e) H. Tsuji, K.
Sato, Y. Sato, E. Nakamura, Chem. Asian J. 2010, 5, 1294 – 1297.
Angew. Chem. Int. Ed. 2015, 54, 1 – 6
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!

.
Angewandte
Communications
Communications
Phosphorus Heterocycles
Phosphole stained: The combination of
an electron-accepting benzophosphole
oxide with an electron-donating (diphe-
nylamino)phenyl group led to a fluores-
cent compound with high fluorescence
quantum yields. The benzophosphole
oxide exhibited a change in fluorescence
emission as a function of the solvent
polarity, and was used to stain adipo-
cytes, thus allowing discrimination of the
polarity of subcellular compartments
based on fluorescence.
E. Yamaguchi, C. Wang, A. Fukazawa,*
M. Taki, Y. Sato, T. Sasaki, M. Ueda,
N. Sasaki, T. Higashiyama,*
S. Yamaguchi*
&&&& — &&&&
Environment-Sensitive Fluorescent
Probe: A Benzophosphole Oxide with an
Electron-Donating Substituent
6
www.angewandte.org
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2015, 54, 1 – 6
These are not the final page numbers!