Environment-sensitive fluorophore emitting in protic environments

Article abstract of DOI:10.1021/ol062490r

(Diagram presented) The unusual fluorescence properties of 8-methoxy-4-methyl-2H-benzo[g]chromen-2-one (1) are described. The fluorophore 1 is almost nonfluorescent in aprotic solvent (e.g., fluorescence quantum yield Φ_f < 0.0003 in n-hexane), whereas it strongly fluoresces at long wavelengths (>450 nm) in protic solvent (e.g., Φ_f = 0.21 in methanol). The fluorophore 1 also shows good applicability in developing a new fluorogenic (fluorescent "off-on") sensor.

Full text of DOI:10.1021/ol062490r

ORGANIC
LETTERS
2
006
Vol. 8, No. 25
869-5872
Environment-Sensitive Fluorophore
Emitting in Protic Environments
5
Seiichi Uchiyama,*,† Kazuyuki Takehira, Toshitada Yoshihara, Seiji Tobita, and
‡
§
§
Tomohiko Ohwada*,
†
Graduate School of Pharmaceutical Sciences, The UniVersity of Tokyo, 7-3-1 Hongo,
Bunkyo-ku, Tokyo 113-0033, Japan, Satellite Venture Business Laboratory, Gunma
UniVersity, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515, Japan, and Department of
Chemistry, Gunma UniVersity, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515, Japan
seiichi@mol.f.u-tokyo.ac.jp; ohwada@mol.f.u-tokyo.ac.jp
Received October 9, 2006
ABSTRACT
The unusual fluorescence properties of 8-methoxy-4-methyl-2H-benzo[g]chromen-2-one (1) are described. The fluorophore 1 is almost
nonfluorescent in aprotic solvent (e.g., fluorescence quantum yield < 0.0003 in n-hexane), whereas it strongly fluoresces at long wavelengths
0.21 in methanol). The fluorophore 1 also shows good applicability in developing a new fluorogenic
Φ
f
(
(
>450 nm) in protic solvent (e.g.,
fluorescent “off on”) sensor.
f
Φ )
−
Fluorogenic (fluorescent “off-on”) molecules have been
fluoresces in a hydrophobic environment, transforms an
external stimulus (input) into fluorescence (output) by way
of a decrease in the local hydrophilicity of the macro-
molecules.
Along this line, it would be necessary to have environ-
ment-sensitive fluorophores with an opposite fluorescence
characteristic, i.e., emitting in a hydrophilic medium when
transforming an increase in the local hydrophilicity of
1
2
widely used as sensors and molecular devices. Recently, a
new type of fluorogenic sensor/device was developed by the
introduction of an environment-sensitive fluorophore into
stimulus-responsive macromolecules such as protein and
3
synthetic polymers. In these systems, an environment-
4
5
sensitive fluorophore, benzofurazan or dansylamine, which
†
The University of Tokyo.
Satellite Venture Business Laboratory, Gunma University.
Department of Chemistry, Gunma University.
‡
(3) (a) Walkup, G. K.; Imperiali, B. J. Am. Chem. Soc. 1996, 118, 3053-
3054. (b) Deo, S.; Godwin, A. J. Am. Chem. Soc. 2000, 122, 174-175. (c)
Uchiyama, S.; Matsumura, Y.; de Silva, A. P.; Iwai, K. Anal. Chem. 2003,
75, 5926-5935. (d) Wada, A.; Mie, M.; Aizawa, M.; Lahoud, P.; Cass, A.
E. G.; Kobatake, E. J. Am. Chem. Soc. 2003, 125, 16228-16234. (e)
Uchiyama, S.; Kawai, N.; de Silva, A. P.; Iwai, K. J. Am. Chem. Soc. 2004,
126, 3032-3033. (f) Uchiyama, S.; Matsumura, Y.; de Silva, A. P.; Iwai,
K. Anal. Chem. 2004, 76, 1793-1798. (g) Iwai, K.; Matsumura, Y.;
Uchiyama, S.; de Silva, A. P. J. Mater. Chem. 2005, 15, 2796-2800.
(4) (a) Uchiyama, S.; Santa, T.; Imai, K. J. Chem. Soc., Perkin Trans. 2
1999, 2525-2532. (b) Uchiyama, S.; Santa, T.; Okiyama, N.; Fukushima,
T.; Imai, K. Biomed. Chromatogr. 2001, 15, 295-318. (c) Ikeda, H.;
Murayama, T.; Ueno, A. Org. Biomol. Chem. 2005, 3, 4262-4267.
(5) Li, Y.-H.; Chan, L.-M.; Tyer, L.; Moody, R. T.; Himel, C. M.;
Hercules, D. M. J. Am. Chem. Soc. 1975, 97, 3118-3126.
§
(1) (a) de Silva, A. P.; Gunaratne, H. Q. N.; Gunnalaugsson, T.; Huxley,
A. J. M.; McCoy, C. P.; Rademacher, J. T.; Rice, T. E. Chem. ReV. 1997,
9
7, 1515-1566. (b) Fabbrizzi, L.; Licchelli, M.; Pallavicini, P. Acc. Chem.
Res. 1999, 32, 846-853. (c) Wiskur, S. L.; Ait-Haddou, H.; Lavigne, J. J.;
Anslyn, E. V. Acc. Chem. Res. 2001, 34, 963-972. (d) Callan, J. F.; de
Silva, A. P.; Magri, D. C. Tetrahedron 2005, 61, 8551-8588.
(
2) (a) Balzani, V.; Credi, A.; Venturi, M. Molecular DeVices and
Machines; Wiley-VCH: Weinheim, 2003. (b) de Silva, A. P.; McClenaghan,
N. D. Chem.-Eur. J. 2004, 10, 574-586. (c) Leigh, D. A.; Morales, M.
AÄ . F.; P e´ rez, E. M.; Wong, J. K. Y.; Saiz, C. G.; Slawin, A. M. Z.;
Carnichael, A. J.; Haddleton, D. M.; Brouwer, A. M.; Buma, W. J.; Wurpel,
G. W. H.; Le o´ n, S.; Zerbetto, F. Angew. Chem., Int. Ed. 2005, 44, 3062-
3
067.
1
0.1021/ol062490r CCC: $33.50
© 2006 American Chemical Society
Published on Web 11/16/2006

Table 1. Photophysical Properties of 1: Fluorescence Quantum Yield (Φ
Absorption Coefficients (ꢀ), Maximum Emission Wavelength (λem), Fluorescence Lifetime (τ
Crossing (Φisc), and Internal Conversion (Φic
f
), Maximum Absorption Wavelength (λabs), Molar
f
), Quantum Yields of Intersystem
)
solvent
Ra
Db
Φf
λabs (nm)
ꢀ (M-1 cm-1
)
λem (nm)
τf (ns)
Φiscc
Φic
n-hexane
0.00
0.00
0.19
0.44
0.83
0.93
(1.17)f
1.9
6.0
37.5
4.8
24.6
32.7
70.7
<0.0003d
0.0006
0.0071
0.011
0.13
337
340
340
344
343
343
345
17500
17800
17600
18900
18600
18600
17500
NDd
435
453
443
464
469
500
NDd
0.066
0.35
0.57
4.2
0.021
0.051
0.071
0.31
0.33
0.34
0.98
0.95
0.92
0.68
0.54
0.45
0.56
ethyl acetate
acetonitrile
chloroform
ethanol
methanol
water-methanole
0.21
0.27
6.7
9.2
0.17
(4:1, v/v)
trifluoroethanol
1.51
26.5
0.33
344
18000
496
13
0.41
0.26
a
Hydrogen-bond donor acidity of solvent (ref 15). Dielectric constant of solvent. c Determined by using the ET value (19 000 cm ) in a mixture of
b
-1
methanol-ethanol (1:1) at 77 K. Below the detection limit. ND: Fluorescence signal was too weak to be detected. e Insoluble in 100% water. f Value for
water.
d
macromolecules (e.g., due to unfolding) into a fluorescence
of 1 to its environment. The fluorophore 1 is nonfluorescent
(Φ < 0.0003) in n-hexane but strongly fluorescent (Φ
0.27) in aqueous solution. This increase in the Φ value of
1 is well correlated with the hydrogen-bond donor acidity
(R) of the solvents used rather than with the dielectric
constant (D, see Table 1). Therefore, it can be concluded
that the fluorescence of 1 requires the hydrogen bonding
between the solvent as a donor and 1 (essentially, its carbonyl
group) as an acceptor; i.e., the fluorophore 1 strongly emits
specifically in a protic environment.
6
signal. However, only acridine, pyrene-3-carboxaldehyde
f
f
)
7
8
(
PCA), and 7-methoxy-4-methylcoumarin (MMC) were
f
reported as fluorophores with this characteristic and the
applicability of these fluorophores is limited because of
1
5
9
fluorescence quenching by protonation, difficulty of struc-
10,11
12
tural modification,
we propose the structure 1 (8-methoxy-4-methyl-2H-benzo-
g]chromen-2-one) as a novel environment-sensitive fluo-
or short emission wavelengths. Here,
[
rophore which strongly emits in the visible wavelength
region, specifically in a protic environment.
The fluorophore 1 was designed by referring the chemical
structures of the conventional fluorophores (i.e., acridine,
PCA, and MMC). The structural key points in 1 are the
lactone ring and the absence of a strong electron-donating
substituent such as a dimethylamino group. The former can
provide the desired sensitivity to environments by involving
8
an n f π* state in excited 1. On the other hand, the latter
1a
can control the intramolecular charge-transfer (ICT) character
of the fluorophore to be moderate, otherwise fluorescence
quenching would be observed in hydrophilic environments
as seen for benzofurazan and dansylamine.
The fluorophore 1 was synthesized by the simple con-
densation of 7-methoxy-2-naphthol and ethyl acetoacetate
Figure 1. Representative absorption (black) and fluorescence
in the presence of sulfuric acid (Scheme 1).1
3,14
Then, the
(
colored) spectra of 1 (10 µM); in acetonitrile (red), ethanol (purple),
methanol (black and blue), and water-methanol (4:1, v/v) (green).
Excitation: λabs
.
Scheme 1. Synthesis of the New Fluorophore 1
The maximum emission wavelength (λem) of 1 is also
sensitive to the environment (Table 1). The longer emission
wavelength in a hydrophilic solvent (e.g., 469 nm in
methanol) compared with that in a hydrophobic solvent (e.g.,
4
35 nm in ethyl acetate) is due to an ICT character of the
(
6) Kellmann, A. J. Phys. Chem. 1977, 81, 1195-1198.
(7) Kalyanasundaram, K.; Thomas, J. K. J. Phys. Chem. 1977, 81, 2176-
fluorescence characteristics of 1 were examined in various
solvents. Table 1 summarizes the absorption and fluorescence
characteristics of 1, and Figure 1 displays representative
spectra. These results clearly demonstrate high sensitivity
2
180.
(8) de Melo, J. S. S.; Becker, R. S.; Ma c¸ anita, A. L. J. Phys. Chem.
994, 98, 6054-6058.
9) Nakamaru, K.; Niizuma, S.; Koizumi, M. Bull. Chem. Soc. Jpn. 1969,
42, 255-277.
1
(
5870
Org. Lett., Vol. 8, No. 25, 2006

1a
excited state. In a mixture of water and methanol (4:1, v/v),
the maximum emission wavelength reached 500 nm. This
long emission wavelength and the large Stokes shift (8900
cm ) are favorable for biorelevant applications. It should
also be noted that 1 is stable even under severe conditions.
No decomposition (i.e., photolysis or hydrolysis) was
observed during excitation (345 nm) in an aqueous basic
solution of pH 9 at 60 °C.
Scheme 2. Synthesis of the Fluorescent Monomer 2
-
1
Table 1 also lists the τ , Φisc, and Φic values of 1 in various
f
solvents.16 In n-hexane, internal conversion was observed
almost exclusively as the relaxation pathway of excited 1.
The efficiency of the internal conversion (Φic) decreased as
3c
behavior opposite to the previous report using benzofurazan ).
The fluorescent monomer 2 was synthesized (Scheme 2),21
and subsequently, the random copolymer 3 (Figure 2a) was
f
that of the fluorescence (Φ ) increased in protic solvents (e.g.,
ethanol, methanol, and water-methanol (4:1)). These data
indicate that internal conversion is the dominant pathway
competing with fluorescence in 1, and protic solvents reduce
17
the contribution of the former process. Such a solvent effect
on the relaxation pathways of 1 is, interestingly, different
from that for acridine6
,18
or PCA, in which inter-
19
system crossing is the main competing process with fluo-
rescence.
Finally, we present a new fluorescent polymeric thermom-
eter emitting at lower temperature as an example of the
potential applications of the new fluorophore.20 We have
recently reported sensitive fluorescent thermometers based
on incorporation of an environment-sensitive fluorophore into
3c
a thermoresponsive poly(N-alkylacrylamide). With heating
of the thermometer in aqueous solution, water molecules
become separate from nanospaces near the main chain of
the polymer, and the fluorophore senses the change in the
local environment. Thus, the fluorophore 1 should afford a
fluorescent thermometer emitting at lower temperature (i.e.,
(
10) The fluorescence signal from the compound bearing the PCA
Figure 2. Fluorescent polymeric thermometer emitting at lower
temperature as an example of potential applications of 1. (a)
Chemical structure of the random copolymer 3. (b) Fluorescence
spectra of 3 (0.01 w/v %) in buffer (pH 4) in different temperatures.
The high solubility of 3 in water is due to the ionizable acrylamide
units. The response to temperature change was reversible. Excita-
tion: 345 nm. (c) Relationship (b) between fluorescence intensity
structure is dramatically reduced if the unstable aldehyde group is replaced
by the stable ketone group. See: Armbruster, C.; Knapp, M.; Rechthaler,
K.; Schamschule, R.; Parusel, A. B. J.; K o¨ hler, G.; Wehrmann, W. J.
Photochem. Photobiol. A: Chem. 1999, 125, 29-38. For photolysis of PCA,
see also ref 18.
(11) It is noteworthy that fluorescent nucleobases with the PCA structure
can distinguish types of single nucleotide polymorphism. See: Okamoto,
A.; Kanatani, K.; Saito, I. J. Am. Chem. Soc. 2004, 126, 4820-4827.
(
f
of 3 at 500 nm and temperature. The Φ value at 10 °C was 0.23.
12) The short maximum excitation and emission wavelengths of MMC
(
330 and 392 nm in water, respectively) are not suitable for biorelevant
The open circle (O) indicates the previously reported fluorescence
behavior of the polymeric thermometer containing a benzofurazan
unit as an environment-sensitive fluorophore (see ref 3e).
applications. See: Muthuramu, K.; Ramamurthy, V. J. Photochem. 1984,
2
6, 57-64.
(13) For details of the synthetic procedure, see Supporting Information.
(14) The major product of the reaction was a regioisomer of 1, which
was less sensitive to environments than 1.
15) Kamlet, M. J.; Abboud, J.-L. M.; Abraham, M. H.; Taft, R. W. J.
Org. Chem. 1983, 48, 2877-2887.
16) To determine these values, measurements of the fluorescence
lifetime, optoacoustic signal, and phosphorescence spectra were carried out.
For details of the experimental procedures and results, see Supporting
Information.
obtained.13 The fluorescence behavior of the copolymer 3
at various temperatures in aqueous solution is shown in
Figure 2b,c. As expected, stronger emission was observed
at lower temperature. The blue shift of the maximum
emission wavelength of 3 from 500 nm (at 5 °C) to 471 nm
(at 50 °C) is consistent with the removal of water molecules
from local spaces near the polymeric structure. Random
copolymers prepared from other N-alkylacrylamides also
showed sharp responses to the decrease in temperature (see
Supporting Information). The fluorescent polymeric ther-
mometers prepared in this study are capable of detecting a
small temperature decrease in an aqueous phase.
(
(
(17) The hydrogen bonding may increase an energy gap between the S1
and S2 states in 1 and diminish a “proximity effect”. For a related discussion,
see: de Melo, J. S.; Becker, R. S.; Elisei, F.; Ma c¸ anita, A. L. J. Chem.
Phys. 1997, 107, 6062-6069.
(
18) (a) Noe, L. J.; Degenkolb, E. O.; Rentzepis, P. M. J. Chem. Phys.
1
978, 68, 4435-4438. (b) Kasama, K.; Kikuchi, K.; Yamamoto, S.; Uji-ie,
K.; Nishida, Y.; Kokubun, H. J. Phys. Chem. 1981, 85, 1291-1296.
19) Kumar, C. V.; Chattopadhyay, S. K.; Das, P. K. Photochem.
Photobiol. 1983, 38, 141-152.
20) For reviews on fluorescent thermometers, see: (a) Chandrasekharan,
(
(
N.; Kelly, L. A. In ReViews in Fluorescence 2004; Geddes, C. D., Lakowicz,
J. R., Eds.; Kluwer Academic/Plenum: New York, 2004; Vol. 1, pp 21-
4
7
0. (b) Uchiyama, S.; de Silva, A. P.; Iwai, K. J. Chem. Educ. 2006, 83,
20-727.
(21) The low yield was mainly due to the production of a regioisomer
(yield, 46%) in the first step.
Org. Lett., Vol. 8, No. 25, 2006
5871

In closing, we have reported the unusual fluorescence
properties of the new fluorophore 1, i.e., emitting strongly
in protic environments. The fluorophore 1 also possesses high
stability, a long emission wavelength, a large Stokes shift,
and facility in structural modification. These unique features
are convenient for developments of new fluorogenic sensors
and devices.
Sankyo Co. Ltd.) for his valuable comments and discussions.
T.O. is grateful for a Grant-in-Aid for Scientific Research
(Category S, No. 17109001) from the Japan Society for the
Promotion of Science.
Supporting Information Available: Experimental details
and supplementary figures (PDF). This material is available
free of charge via the Internet at http://pubs.acs.org.
Acknowledgment. S.U. thanks The Sumitomo Founda-
tion for financial support. We thank Dr. J. Kondo (Daiichi
OL062490R
5872
Org. Lett., Vol. 8, No. 25, 2006