New fluorogenic benzothiadiazole and benzoselenadiazole reagents to yield environment-sensitive fluorophores via a reaction with amines

Article abstract of DOI:10.1246/cl.2012.1451

We introduce 7-fluoro-4-N,N-dimethylaminosulfonyl-2,1,3-benzothiadiazole and 7-fluoro-4-N,N-dimethylaminosulfonyl-2,1,3-benzoselenadiazole as new fluorogenic reagents for amines. These reagents are nonfluorescent themselves and can easily react with nonfl

Full text of DOI:10.1246/cl.2012.1451

doi:10.1246/cl.2012.1451
Published on the web November 3, 2012
1451
New Fluorogenic Benzothiadiazole and Benzoselenadiazole Reagents
to Yield Environment-sensitive Fluorophores via a Reaction with Amines
Kyoko Kawamoto and Seiichi Uchiyama*
Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033
(Received July 6, 2012; CL-120720; E-mail: seiichi@mol.f.u-tokyo.ac.jp)
We introduce 7-fluoro-4-N,N-dimethylaminosulfonyl-2,1,3-
benzothiadiazole and 7-fluoro-4-N,N-dimethylaminosulfonyl-
2,1,3-benzoselenadiazole as new fluorogenic reagents for
amines. These reagents are nonfluorescent themselves and can
easily react with nonfluorescent amines to produce environment-
sensitive fluorophores. Herein, we report the synthesis of the
new reagents and the fluorescence properties of the reagents and
their amine derivatives as well as the reactivity of the new
reagents toward amines.
R
R
a (R = F)
b (R = NMe2)
c (R = NHCHCOOH)
N
N
N
N
S
Se
Me
SO2NMe2
SO2NMe2
d (R = Cl)
1a–1d
2a–2d
Figure 1. Chemical structures of benzothiadiazole derivatives
1a-1d and benzoselenadiazole derivatives 2a-2d.
F
F
F
NHAc
F
NH2
NH2
NH2
i, ii
iii
Environment-sensitive fluorophores change their fluores-
cence properties (e.g., maximum emission wavelength, fluores-
cence quantum yield, and fluorescence lifetime) depending on the
polarity of the solvent and the degree of hydrogen bonding with
the solvent molecules. Originally, environment-sensitive fluoro-
phores were utilized for the evaluation of the microenvironment
associated with proteins.1,2 Recently, environment-sensitive
fluorophores have been applied in the development of fluorescent
sensors by incorporating the fluorophores into stimulus-respon-
sive macromolecules, such as proteins and synthetic polymers.3-5
Although different types of environment-sensitive fluorophores
are currently available, new compounds with improved properties
(e.g., high sensitivity, high photostability, and long emission
wavelength) are still in demand. In this letter, we introduce 7-
fluoro-4-N,N-dimethylaminosulfonyl-2,1,3-benzothiadiazole (1a)
and 7-fluoro-4-N,N-dimethylaminosulfonyl-2,1,3-benzoselena-
diazole (2a) (Figure 1) as new fluorogenic reagents for amines;
these are nonfluorescent themselves and produce environment-
sensitive fluorophores in the presence of amines. The synthesis
and photophysical properties of 1a, 2a, and their amine
derivatives and the reactivity of 1a and 2a toward amines are
discussed.
NO2
F
N
N
N
iv
v
vi
1a, 2a
X
X
N
SO2Cl
X = S, Se
Figure 2. Synthesis of 1a and 2a. (i) Fuming HNO3, 0 °C,
1.5 h; (ii) HCl, MeOH, reflux, 5 h (13% in 2 steps); (iii) Fe, HCl,
CH2Cl2, MeOH, rt, 75 min (66%); (iv) X = S, PhNSO, toluene,
reflux, 4.5 h (65%); X = Se, SeO2, EtOH, 80 °C, 1 h (97%); (v)
ClSO3H, 0 °C, 1 h ¼ 150 °C, 2 h (X = S, 92%; X = Se, 93%);
(vi) Me2NH, CH3CN, CH2Cl2, 0 °C, 70 min ¼ rt, 40 min
(X = S, 55%); 0 °C, 15 min ¼ rt, 45 min (X = Se, 45%).
1a
2a
0
0
0
0
1b
2b
The reagents 1a and 2a were synthesized from commer-
cially available 2¤-fluoroacetanilide using a 6-step reaction
(Figure 2). The derivatives of 1a and 2a with N,N-dimethyl-
amine (1b and 2b) and L-alanine (1c and 2c) were also obtained
as model environment-sensitive fluorophores derived from the
new reagents.
The absorption and fluorescence spectra of 1a, 1b, 2a,
and 2b were obtained in n-hexane, ethyl acetate, acetonitrile,
methanol, and water. Fluorescence lifetime measurements
were also performed on 1b and 2b using the same solvents.
Representative absorption and fluorescence spectra of 1a, 1b,
2a, and 2b are provided in Figure 3, and the photophysical
properties of 1b and 2b are summarized in Table 1. As
illustrated in Figure 3, the reagents 1a and 2a did not exhibit
fluorescence in any of the solvents tested. In contrast, the amine
derivatives 1b and 2b showed remarkable fluorescence when
excited at the maximum absorption wavelength. It should be
noted that the fluorescence of 1b and 2b was strongly dependent
0
0
0
0
300 400 500 600 700 800
300 400 500 600 700 800
Wavelength/nm
Wavelength/nm
Figure 3. Representative spectra of 1a, 1b, 2a, and 2b. The
absorption spectra (30 ¯M) were measured in ethyl acetate
(black). The fluorescence spectra (1 ¯M for 1b; 5 ¯M for 2b)
were measured at the excitation wavelength of -abs in ethyl
acetate (orange), methanol (blue), and water (green).
on the nature of the solvents: whereas efficient fluorescence was
observed in an apolar and aprotic solvent, such as n-hexane, the
fluorescence was dramatically quenched in a polar and protic
solvent, such as water. In addition, the maximum emission
wavelength shifted bathochromically with increasing solvent
polarity. The variation in the fluorescence quantum yields of
1b and 2b in different solvents was much larger than that of
conventional environment-sensitive fluorophores, such as dan-
Chem. Lett. 2012, 41, 1451-1452
© 2012 The Chemical Society of Japan
www.csj.jp/journals/chem-lett/

1452
Table 1. Photophysical properties of 1b and 2b at 25 °C:
maximum absorption wavelength (-abs), maximum emission
wavelength (-em), fluorescence quantum yield (Φf), fluorescence
lifetime (¸f), fluorescence rate constant (kf), and nonradiative
rate constant (knr)
sensitive fluorophore, 7-N,N-dimethylamino-4-N,N-dimethyl-
aminosulfonyl-2,1,3-benzoxadiazole (Figure S1 and Table S1).13
However, the photostability of 1b was determined to be
approximately 10 times higher than that of the corresponding
benzoxadiazole (Figure S2),13 demonstrating the potential ap-
plicability of 1a for bioimaging.
-
-
¸
f
kf
knr
abs
em
Comp. Solvent
1b n-Hexane
Φf
¹1
¹1
/nm /nm
/ns /107 s /107 s
Finally, the reactivity of fluorinated reagents 1a and 2a with
amines was compared with that of the corresponding chlorinated
compounds 1d11 and 2d9 (Figure 1). L-Alanine was adopted as
the model amine, and the photophysical properties of L-alanine
derivatives 1c and 2c are provided in Figure S3 and Table S2.13
In this study, the production of 1c and 2c was monitored using
HPLC-UV. The results demonstrate that the fluorinated reagents
1a and 2a (1 mM) were successfully derivatized with excess
L-alanine (30 mM) in a mixture of borate buffer (pH 9.2) and
acetonitrile (1:1, v/v) at 70 °C after reacting for 10 h (yield: 74-
79%). In contrast, almost no reaction was observed between the
chlorinated compounds 1d and 2d and L-alanine under these
conditions (<0.35%). The reaction rates of 1a and 1b with
L-alanine were estimated to be at least 200 times larger than
those of 1d and 2d. The difference in reactivity between
fluorinated and chlorinated reagents might be due to the reaction
mechanism: the intermediate state of this reaction involves a
Meisenheimer complex in which the fluorine atom is less
repulsive to nucleophilic alanine than the chlorine atom.12
Overall, we conclude that the new fluorogenic reagents 1a and
2a are sufficiently reactive toward amines. We are currently
working on developing macromolecular fluorescent sensors
utilizing these fluorogenic reagents.
437 520 0.43
Ethyl acetate 444 570 0.33
Acetonitrilea 445 583 0.20
20.9
19.7
13.3
3.9
2.0
1.7
1.5
1.4
1.0
1.3
1.2
1.0
0.9
0.6
2.8
3.4
6.0
Methanol
Water
2b n-Hexane
446 597 0.053
448 641 0.0044 0.45
476 573 0.19
24
>200
5.7
14.3
6.4
4.7
1.4
Ethyl acetate 479 628 0.077
Acetonitrilea 481 645 0.046
15
20
72
Methanol
Water
481 655 0.013
485 688 0.0011 0.20
>500
aMolar absorption coefficients of 1b and 2b in acetonitrile are
6900 and 6600 M¹1 cm¹1, respectively.
sylamine (cf. Φf = 0.31 in n-hexane and Φf = 0.05 in water)6
and 4-methylamino-7-nitro-2,1,3-benzoxadiazole (cf. Φf = 0.09
in n-hexane and Φf = 0.028 in water).7
Because 1b and 2b bear an electron-donating dimethyl-
amino group and an electron-withdrawing aromatic ring
(i.e., dimethylaminosulfonylbenzothiadiazole or -benzoselenadi-
azole), the first excited singlet (S1) state of 1b and 2b exhibits
intramolecular charge-transfer characteristics. Thus, the absorp-
tion energies required for the transition of 1b and 2b from the
ground (S0) state to the S1 state are lower than those of 1a and
2a, which lack an electron-donating substituent. Similarly, it can
be concluded that the bathochromic shift in the maximum
emission wavelength of 1b and 2b with increasing solvent
polarity is caused by the intramolecular charge-transfer character
of the S1 states.8
We thank JST for the “Development of Advanced Measure-
ment and Analysis System” grant.
References and Notes
1
2
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solvent, the formation of a hydrogen-bonding network between
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have similar polarity (cf. the dielectric constants of methanol and
acetonitrile were 32.7 and 37.5, respectively). Thus, both the
polarity and hydrogen-bonding ability of the solvent influence
the fluorescence properties of 1b and 2b.
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It should be noted that the fluorescence properties of 1b are
almost identical to those of a well-established environment-
Chem. Lett. 2012, 41, 1451-1452
© 2012 The Chemical Society of Japan
www.csj.jp/journals/chem-lett/