Environment-sensitive fluorophores with benzothiadiazole and benzoselenadiazole structures as candidate components of a fluorescent polymeric thermometer

Article abstract of DOI:10.1002/chem.201200597

An environment-sensitive fluorophore can change its maximum emission wavelength (λ_em), fluorescence quantum yield (φ_f), and fluorescence lifetime in response to the surrounding environment. We have developed two new intramolecular charge-transfer-type environment-sensitive fluorophores, DBThD-IA and DBSeD-IA, in which the oxygen atom of a well-established 2,1,3-benzoxadiazole environment-sensitive fluorophore, DBD-IA, has been replaced by a sulfur and selenium atom, respectively. DBThD-IA is highly fluorescent in n-hexane (φ_f=0. 81, λ_em=537 nm) with excitation at 449 nm, but is almost nonfluorescent in water (φ_f=0.037, λ_em=616 nm), similarly to DBD-IA (φ_f=0.91, λ_em=520 nm in n-hexane; φ_f=0.027, λ_em=616 nm in water). A similar variation in fluorescence properties was also observed for DBSeD-IA (φ_f=0.24, λ_em=591 nm in n-hexane; φ_f=0.0046, λ_em=672 nm in water). An intensive study of the solvent effects on the fluorescence properties of these fluorophores revealed that both the polarity of the environment and hydrogen bonding with solvent molecules accelerate the nonradiative relaxation of the excited fluorophores. Time-resolved optoacoustic and phosphorescence measurements clarified that both intersystem crossing and internal conversion are involved in the nonradiative relaxation processes of DBThD-IA and DBSeD-IA. In addition, DBThD-IA exhibits a 10-fold higher photostability in aqueous solution than the original fluorophore DBD-IA, which allowed us to create a new robust molecular nanogel thermometer for intracellular thermometry. Sensitive fluorophores: Two new environment-sensitive fluorophores, DBThD-IA and DBSeD-IA (see figure), are reported. Their photophysical and photochemical properties have been investigated by UV/Vis absorption, fluorescence, and phosphorescence spectroscopy, analysis of the fluorescence lifetimes and optoacoustic signals, and by performing photolysis experiments. DBThD-IA has successfully been used to develop a photostable fluorescent nanogel thermometer. Copyright

Full text of DOI:10.1002/chem.201200597

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DOI: 10.1002/chem.201200597
Environment-Sensitive Fluorophores with Benzothiadiazole and
Benzoselenadiazole Structures as Candidate Components of a Fluorescent
Polymeric Thermometer
Seiichi Uchiyama,*[a] Kohki Kimura,[b] Chie Gota,[a] Kohki Okabe,[a] Kyoko Kawamoto,[a]
Noriko Inada,[c] Toshitada Yoshihara,[b] and Seiji Tobita*[b]
Abstract: An environment-sensitive
fluorophore can change its maximum
emission wavelength (lem), fluores-
cence quantum yield (Ff), and fluores-
cence lifetime in response to the sur-
rounding environment. We have devel-
oped two new intramolecular charge-
transfer-type environment-sensitive flu-
orophores, DBThD-IA and DBSeD-
IA, in which the oxygen atom of
a well-established 2,1,3-benzoxadiazole
fluorescent in water (Ff =0.037, lem
=
bonding with solvent molecules accel-
erate the nonradiative relaxation of the
excited fluorophores. Time-resolved
optoacoustic and phosphorescence
measurements clarified that both inter-
system crossing and internal conversion
are involved in the nonradiative relaxa-
tion processes of DBThD-IA and
DBSeD-IA. In addition, DBThD-IA
exhibits a 10-fold higher photostability
in aqueous solution than the original
fluorophore DBD-IA, which allowed
us to create a new robust molecular
nanogel thermometer for intracellular
thermometry.
616 nm), similarly to DBD-IA (Ff =
0.91, lem =520 nm in n-hexane; Ff =
0.027, lem =616 nm in water). A similar
variation in fluorescence properties
was also observed for DBSeD-IA (Ff =
0.24, lem =591 nm in n-hexane; Ff =
0.0046, lem =672 nm in water). An in-
tensive study of the solvent effects on
the fluorescence properties of these
fluorophores revealed that both the po-
larity of the environment and hydrogen
environment-sensitive
fluorophore,
DBD-IA, has been replaced by a sulfur
and selenium atom, respectively.
DBThD-IA is highly fluorescent in n-
hexane (Ff =0.81, lem =537 nm) with
excitation at 449 nm, but is almost non-
Keywords: chromophores · fluores-
cence · photochemistry · photophy-
sics · sensors
Introduction
Environment-sensitive fluorophores exhibit different fluo-
rescence properties (e.g., maximum emission wavelength,
fluorescence quantum yield, and fluorescence lifetime) de-
pending on their surroundings. In previous studies, prodan,[1]
ANS–Na,[2] dansylamine,[3] NBD-NHMe (nitrobenzofura-
zan),[4] and Nile red[5] (Figure 1, the chemical names are pro-
[a] Dr. S. Uchiyama, C. Gota, Dr. K. Okabe, K. Kawamoto
Graduate School of Pharmaceutical Sciences
The University of Tokyo
Figure 1. Chemical structures of representative examples of environment-
sensitive fluorophores.
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan)
Fax : (+81)3-58414768
E-mail: seiichi@mol.f.u-tokyo.ac.jp
vided in the Experimental Section) were used as representa-
tive environment-sensitive fluorophores. Most conventional
environment-sensitive fluorophores, including these repre-
sentatives, exhibit weaker emission at longer wavelengths in
more polar and protic environments, but a few examples dis-
play different behavior.[6] Thus, environment-sensitive fluo-
rophores can be used for evaluating the microenvironment
near a three-dimensional structure, such as a protein,[7]
DNA,[8] micelle,[9] or silica.[10] In addition, the dynamic con-
formational changes of a protein that are induced by exter-
nal stimuli (e.g., heat,[11] voltage,[12] or inhibitors[13]), protein–
[b] K. Kimura, Dr. T. Yoshihara, Prof. S. Tobita
Department of Chemistry and Chemical Biology
Graduate School of Engineering, Gunma University
1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515 (Japan)
Fax : (+81)277-30-1213
E-mail: tobita@gunma-u.ac.jp
[c] Dr. N. Inada
Graduate School of Biological Sciences
Nara Institute of Science and Technology
8916-5 Takayama-cho, Ikoma, Nara 630-0192 (Japan)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201200597.
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FULL PAPER
protein[14] (or protein–peptide[15]) interactions, and the inser-
tion of a protein into a cell membrane[16] can also be moni-
tored by using environment-sensitive fluorophores.[17]
methanol, [D1]methanol (CH3OD), acetonitrile, DMSO,
water, and deuterium oxide to investigate the sensitivity of
the fluorophores to variations in their environment. Readers
can refer to our previous research in which the same set of
solvents were used to investigate the photophysical proper-
ties of DBD-IA.[32] The fluorescence properties of DBThD-
IA and DBSeD-IA in mixed solvents of 1,4-dioxane and
water were also measured and compared with those of con-
ventional environment-sensitive fluorophores. The photosta-
bilities of DBThD-IA, DBSeD-IA, and DBD-IA were mea-
sured in selected nonaqueous and aqueous solvents because
bioimaging with a fluorescence microscope is a potential ap-
plication of these environment-sensitive fluorophores. In ad-
dition, time-resolved optoacoustic[35] and phosphorescence
experiments were performed for DBThD-IA, DBSeD-IA,
DBD-IA, and their derivatives sensitized by an iridium com-
plex to determine the efficiencies of intersystem crossing
and internal conversion in the relaxation processes.
Recently, another use for environment-sensitive fluoro-
phores was reported for new fluorescent sensing sys-
tems[18–20] in which an environment-sensitive fluorophore is
covalently attached to a stimulus-responsive macromolecule
(e.g., protein, peptide, or synthetic polymer). In the pioneer-
ing work reported by Walkup and Imperiali,[21] a zinc-bind-
ing peptide labeled with dansylamine functioned as a fluores-
cent zinc ion sensor: The environment-sensitive fluorophore
transformed the microenvironmental change induced by the
binding event between the peptide and the zinc ion into
a fluorescence signal. Within a decade, this type of fluores-
cent sensor was intensively developed to target various ions
(calcium,[22] copper,[23] lead,[24] sulfate,[25] and phosphate[26]
)
and small molecules (glucose,[27] maltose,[28] and gluta-
mine[29]). A fluorescent macromolecular sensor for Pi (inor-
ganic phosphate) has been used to study the kinetics of ATP
and GTP hydrolysis.[30]
Finally, one of the new fluorophores, DBThD-IA, was
used as an environment-sensitive fluorophore to construct
a fluorescent polymeric thermometer because it exhibits
high photostability compared with DBD-IA used in previous
A substituted 2,1,3-benzoxadiazole (benzofurazan)[31] is
one fluorophore that is very sensitive to changes in the po-
larity and the hydrogen-bonding ability of a solvent. Al-
though NBD-NHMe[4b] (Figure 1) is a more common and
readily available environment-sensitive fluorophore with
a benzofurazan structure, we used DBD-IA[32] (Figure 2) as
studies.[32–34]
A
fluorescent monomer, DBThD-AA
(Figure 3), and a fluorescent nanogel thermometer com-
posed of DBThD-IA units were
prepared. Studies on living cells
revealed that this new fluores-
cent nanogel thermometer is re-
markably resistant to photo-
bleaching and therefore suit-
AHCTUNGTERGaNNUN ble for long-term monitoring
of intracellular temperatures.
Thus, DBThD-IA can replace
DBD-IA, especially in cases in
which the environment-sensi-
tive-fluorophore-containing flu-
Figure 3. Chemical structure of
new environment-sensitive
fluorescent monomer, DBThD-
AA.
a
orescent
macromolecular
Figure 2. Chemical structures of DBD-IA, DBThD-IA, and DBSeD-IA.
sensor has been designed for
biological experiments accom-
panied by light irradiation.
a more sensitive fluorophore in previous studies for the de-
velopment of thermoresponsive polymer-based fluorescent
thermometers.[33] Owing to the high sensitivity of DBD-IA
to its surroundings, a fluorescent polymeric thermometer
Results
composed of a thermoresponsive poly(N-isopropylacryl
ACHTUNGERTNaNUNG m-
A
Synthesis of DBThD-IA and DBSeD-IA: DBThD-IA and
DBSeD-IA were synthesized according to Scheme 1. The
2,1,3-benzthiodiazole ring was obtained by the reaction be-
tween 1-chloro-2,3-diaminobenzene with N-thionylaniline,
whereas the 2,1,3-benzoselenadiazole ring was constructed
from the same diaminobenzene and selenium dioxide. A di-
methylsulfamoyl group was introduced into the aromatic
rings by using chlorosulfuric acid and an equimolar amount
of dimethylamine. The chloro group was then replaced by
an amino group by using N,N’-dimethylethylenediamine and
subsequent acylation with isobutyric anhydride afforded
DBThD-IA and DBSeD-IA.
a 13.3-fold increase in fluorescence when the temperature of
the aqueous solution increased from 29 to 378C. A very
small change (0.3–0.58C) in the temperature inside living
cells can be monitored by using a fluorescent polymeric
thermometer created in the same manner.[34]
In this paper, we report on disubstituted benzothiadiazole
and benzoselenadiazole derivatives (DBThD-IA and
DBSeD-IA, shown in Figure 2) as new environment-sensi-
tive fluorophores. The absorption and fluorescence spectra
as well as the fluorescence lifetimes of the synthesized
DBThD-IA and DBSeD-IA were measured in n-hexane,
1,4-dioxane, ethyl acetate, ethanol, 2,2,2-trifluoroethanol,
Chem. Eur. J. 2012, 18, 9552 – 9563
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S. Uchiyama, S. Tobita et al.
Scheme 1. Synthesis of DBThD-IA and DBSeD-IA. Reagents and condi-
tions: i) PhNSO, toluene, reflux, 3 h (86%); ii) ClSO3H, 08C, 1.5 h!
1508C, 2.5 h (79%); iii) Me2NH, H2O, MeCN, CH2Cl2, RT, 30 min
(98%); iv) N,N’-dimethylethylenediamine, MeCN, 808C, 1 h (>99%);
v) isobutyric anhydride, TEA, MeCN, RT, 1 h (78%); vi) SeO2, EtOH,
808C, 30 min (99%); vii) ClSO3H, 08C, 1 h!1508C, 3 h (66%); viii)
Me2NH, H2O, MeCN, CH2Cl2, RT, 30 min (85%); ix) N,N’-dimethylethACTHNUTRGENUGyN l-
ACHTUNGTRENNUNG
Absorption and fluorescence properties of DBThD-IA and
DBSeD-IA: The absorption and fluorescence spectra of
DBThD-IA and DBSeD-IA were recorded in 11 solvents of
various polarities and hydrogen-bonding abilities, that is, n-
hexane, 1,4-dioxane, ethyl acetate, ethanol, 2,2,2-trifluoro-
Figure 4. Representative absorption (black, 30 mm) and fluorescence (col-
ored, 5 mm, excited at labs) spectra (top) and fluorescence decay curves
(5 mm, at lem with excitation at 456 nm) (bottom) of a) DBThD-IA and
b) DBSeD-IA at 258C in 1,4-dioxane (orange), acetonitrile (black and
red), ethanol (purple), methanol (blue), and water (green).
ACHTUNGTRENNUNGethanol, methanol, [D1]methanol (CH3OD), acetonitrile,
DMSO, water, and deuterium oxide. Representative absorp-
tion and fluorescence spectra of DBThD-IA and DBSeD-
IA are shown in Figure 4, and the maximum absorption
wavelengths, molar absorption coefficients, maximum emis-
sion wavelengths, and fluorescence quantum yields are listed
in Table 1. The fluorescence lifetimes of DBThD-IA and
DBSeD-IA were measured in the same solvents. Represen-
tative fluorescence decay curves are also shown in Figure 4.
Each fluorescence decay curve is well fitted by a single-ex-
ponential curve and the fluorescence lifetimes are listed in
Table 1. The fluorescence and nonradiative rate constants
were calculated and are also listed in Table 1. The results in-
dicate that the fluorescence properties, that is, the maximum
emission wavelengths, fluorescence quantum yields, and
fluorescence lifetimes of DBThD-IA and DBSeD-IA are af-
fected by the solvent, whereas the absorption properties,
that is, the maximum absorption wavelengths and molar ab-
sorption coefficients, are relatively independent of solvent.
In less polar or less protic solvents, the fluorescence quan-
tum yields of both fluorophores are higher and their fluores-
cence lifetimes are longer. Comparison of the fluorescence
properties of DBThD-IA and DBSeD-IA in the same sol-
vent revealed that the former fluoresces more strongly at
a shorter wavelength, as indicated in Table 1.
Absorption and fluorescence properties of conventional en-
vironment-sensitive fluorophores: The absorption and fluo-
rescence spectra of prodan, ANS-Na, dansylamine, NBD-
NHMe, Nile red, and DBD-IA were recorded in binary mix-
tures of 1,4-dioxane and water (see Figure S1 in the Sup-
porting Information). For comparison, the absorption and
fluorescence spectra of DBThD-IA and DBSeD-IA were
also recorded in mixtures of 1,4-dioxane and water. The
maximum absorption wavelengths, maximum emission
wavelengths, and fluorescence quantum yields are presented
in Table 2.
Photostabilities of DBThD-IA, DBSeD-IA, and DBD-IA:
To evaluate the stabilities of DBThD-IA, DBSeD-IA, and
DBD-IA in the excited state, photolysis experiments were
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Environment-Sensitive Fluorophores
FULL PAPER
Table 1. Photophysical properties of DBThD-IA and DBSeD-IA in various solvents at 258C: Maximum absorption wavelength (labs), molar absorption
coefficient (e), maximum emission wavelength (lem), fluorescence quantum yield (Ff), fluorescence lifetime (tf), fluorescence rate constant (kf), and non-
radiative rate constant (knr).
Compound
DBThD-IA
Solvent
D[a]
a[b]
labs [nm]
e [mꢀ1 cmꢀ1
]
lem [nm]
Ff
tf [ns]
kf [107 sꢀ1
]
knr [107 sꢀ1
]
n-hexane
1.88
2.24
6.02
0.00
0.00
0.00
0.83
1.51
0.93
449
448
449
448
443
447
447
448
455
449
449
489
485
484
482
482
482
481
484
485
486
485
8100
7700
7600
8000
9500
7900
7800
8000
8100
8300
8200
7000
7000
7100
7200
8400
7700
7600
7700
7000
7700
8300
537
570
573
581
590
589
590
586
598
616
619
591
624
629
638
645
644
648
639
653
672
682
0.81
0.73
0.68
0.39
0.13
0.27
0.39
0.49
0.40
0.037
0.10
0.24
0.14
22.9
22.6
21.2
15.2
7.0
12.0
16.0
18.6
14.4
1.90
5.65
11.6
6.8
6.3
3.5
1.3
2.5
3.7
5.3
4.6
0.45
1.1
3.5
3.2
3.2
2.6
1.8
2.3
2.4
2.6
2.8
1.9
1.8
2.1
2.1
1.9
1.7
1.4
1.5
1.7
1.7
2.0
1.0
1.4
0.8
1.2
1.5
4.0
12
6.1
3.8
2.7
4.2
51
16
6.6
13
14
27
74
38
26
17
20
220
89
1,4-dioxane
ethyl acetate
ethanol
2,2,2-trifluoroethanol
methanol
24.6
26.5
32.7
31.7[c]
37.5
46.7
80.4
78.3[e]
1.88
2.24
6.02
24.6
26.5
32.7
31.7[c]
37.5
46.7
80.4
78.3[e]
[D1]methanol
acetonitrile
DMSO
0.19
0.00
1.17
water[d]
deuterium oxide[d]
n-hexane
DBSeD-IA
0.00
0.00
0.00
0.83
1.51
0.93
1,4-dioxane
ethyl acetate
ethanol
2,2,2-trifluoroethanol
methanol
[D1]methanol
acetonitrile
DMSO
0.12
0.059
0.018
0.038
0.061
0.089
0.093
0.0046
0.016
0.19
0.00
1.17
water
deuterium oxide
[a] Dielectric constant of the solvent. [b] Hydrogen-bond donor acidity of the solvent from ref. [62]. DBThD-IA and DBSeD-IA behave only as a hydro-
gen-bond acceptor because there is no acidic proton in the structure. [c] See ref. [63] . [d] The photophysical data in this solvent is cited from ref. [32].
[e] See ref. [64].
carried out. Figure 5 shows the changes in the absorption
spectra of DBThD-IA, DBSeD-IA, and DBD-IA in acetoni-
trile following photoirradiation with an ultra-high-pressure
mercury lamp (l>390 nm).[36] The absorption spectrum of
DBThD-IA was largely unchanged following photoirradia-
tion for 20 min, whereas the spectra of DBSeD-IA and
DBD-IA showed photodecomposition under the same con-
ditions. To determine whether the photoreactive state was
a singlet or a triplet, complementary photolysis experiments
were also performed on DBSeD-IA and DBD-IA in de-
served for DBThD-IA, DBSeD-IA, and DBD-IA in all sol-
utions compared with those of the reference solutions (see
Figures S3–S5 in the Supporting Information and also the
b values derived from Equation (5) in the Experimental Sec-
tion and listed in Table 4). This result indicates that a certain
degree of intersystem crossing is involved in the relaxation
processes of DBThD-IA, DBSeD-IA, and DBD-IA. The
phosphorescence spectra of these fluorophores were then re-
corded to determine the dissipated energy from the first
triplet excited (T1) state. Although no phosphorescence was
observed from DBThD-IA, DBSeD-IA, or DBD-IA, the
phosphorescence spectra corresponding to DBThD-IA and
DBD-IA could be obtained from DBThD-BTPSA and
DBD-BTPSA, which have a sensitizing iridium complex,
BTPSA, unit in addition to the fluorescent DBThD-IA or
DBD-IA moiety (Figure 6). These iridium complexes were
obtained by the condensation of BTPSA with the fluores-
cent amine with a benzothiadiazole or benzoxadiazole ring
(see Scheme S1 in the Supporting Information). Iridium
complexes generally show strong phosphorescence, even at
room temperature.[39] In DBThD-BTPSA and DBD-
BTPSA, triplet–triplet energy transfer occurred from the
BTPSA unit to the DBThD-IA and DBD-IA fluorophores.
Because the quantum yields for photodecomposition were
negligible for DBThD-IA, DBSeD-IA, and DBD-IA (see
Table 3), the quantum yields for intersystem crossing (or in-
ternal conversion) could be calculated from the b values ob-
tained from the optoacoustic measurements, the dissipated
energies of the S1 state (determined from the fluorescence
spectra) and the T1 state (determined from the phosphores-
ACHTUNGTRENNUNGaerated acetonitrile and indicated that the photodecomposi-
tion of DBSeD-IA was dramatically suppressed in the ab-
sence of dissolved oxygen (see Figure S2 in the Supporting
Information). The quantum yields for the photodecomposi-
tion were determined in 1,4-dioxane, acetonitrile, methanol,
and a mixture of water and methanol (4:1, v/v) by using
a previously established method with a spectrofluorimeter
and HPLC.[37] As shown in Table 3, the quantum yields were
found to be (1.6–5.9)ꢁ10ꢀ4 for DBThD-IA, (0.8–12)ꢁ10ꢀ3
for DBSeD-IA, and (1.7–3.8)ꢁ10ꢀ3 for DBD-IA.[38] These
results indicate that DBThD-IA is more stable than
DBSeD-IA or DBD-IA under photoirradiation.
Intersystem crossing and internal conversion of DBThD-IA,
DBSeD-IA, and DBD-IA: Intersystem crossing and internal
conversion were investigated to gain an in-depth under-
standing of the relaxation process from the first singlet ex-
cited (S1) state of the fluorophores. First, optoacoustic meas-
urements were performed in acetonitrile, methanol, and
water as solvent; relatively low signal amplitudes were ob-
Chem. Eur. J. 2012, 18, 9552 – 9563
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9555